Compositions and methods for measuring blood glucose levels

Abstract

In some embodiments, the present invention a mutated FAD-GDH protein, wherein the mutated FAD-GDH protein is mutated from a wild-type first species to contain at least one point mutation, wherein the mutated FAD-GDH protein comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of X, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

Claims

1. A mutated Flavoprotein Glucose Dehydrogenase, subunit alpha (FAD-GDH) protein, wherein the mutated FAD-GDH protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39, and comprising at least two substitutions at the positions corresponding to residues 177, 215, 353 or 406 of the SEQ ID NO: 38 or SEQ ID NO: 39, wherein the amino acid at the position corresponding to residue 177 is S, wherein the amino acid at the position corresponding to residue 215 is selected from the group consisting of S, G, H, D and Y, wherein the amino acid at the position corresponding to residue 353 is L, and wherein the amino acid at the position corresponding to residue 406 is selected from the group consisting of L, S, C, T, M, V, Y, N, P, G, Q, A, I, D, H, and E.

2. The mutated FAD-GDH protein of claim 1, wherein the amino acid sequence of the mutated FAD-GDH protein comprises the amino acid sequence set forth in SEQ ID NO: 110 or SEQ ID NO: 111.

3. The mutated FAD-GDH protein of claim 1, wherein the protein exhibits at least a 10% increase in glucose dehydrogenase activity compared to a non-mutated FAD-GDH protein comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39, wherein the protein exhibits at least a 10% increase in selectivity for glucose compared to a non-mutated FAD-GDH protein comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39 or wherein the protein exhibits at least a 10% increase in linearity of current as a function of glucose concentration compared to a non-mutated FAD-GDH protein comprising the amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

4. The mutated FAD-GDH protein of claim 1, wherein the mutated FAD-GDH protein comprises at least two substitutions, wherein the at least two substitutions are selected from the group consisting of N177S, N215S, F353L and F406L.

5. A Flavoprotein Glucose Dehydrogenase alpha subunit (FAD-GDH) protein, wherein the amino acid sequence of the FAD-GDH protein comprises the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39 and comprises N177S, N215S, F353L and F406L substitutions.

6. A mutated Flavoprotein Glucose Dehydrogenase, subunit alpha (FAD-GDH) protein, wherein the mutated FAD-GDH protein comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence set forth in SEQ ID NO: 38 or SEQ ID NO: 39, and comprising a substitution at the position corresponding to residue 406 of the SEQ ID NO: 38 or SEQ ID NO: 39 and at least one substitution at the position corresponding to residues 177, 215 or 353 of the SEQ ID NO: 38 or SEQ ID NO: 39, wherein the amino acid the position corresponding to residue 406 is selected from the group consisting of L, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, H, and E, wherein the amino acid the position corresponding to residue 177 is S, wherein the amino acid the position corresponding to residue 215 is selected from the group consisting of S, G, H, D and Y, and wherein the amino acid the position corresponding to residue 353 is L.

7. An enzyme electrode, configured to measure the amount of glucose in a physiological fluid, comprising the mutated FAD-GDH of claim 5 immobilized onto the electrode, wherein the mutated FAD-GDH is configured to catalyze glucose in the physiological fluid and produce electrons that are transferred to the electrode thereby generating an electrical current, wherein the intensity of the electrical current is indicative of the level of glucose in the physiological fluid.

8. The enzyme electrode of claim 7, wherein the enzyme electrode is configured to perform a single measurement.

9. The enzyme electrode of claim 7, wherein the enzyme electrode is incorporated into a glucose test strip.

10. The enzyme electrode of claim 7, wherein the mutated FAD-GDH is immobilized on the electrode in a conductive matrix or by chemical wiring.

11. The enzyme electrode of claim 10, wherein the conductive matrix is selected from a group consisting of carbon paste, graphite paste, and graphene oxide.

12. The enzyme electrode of claim 7, wherein the enzyme electrode, configured to measure the amount of glucose in a physiological fluid, comprising the mutated FAD-GDH protein immobilized onto the electrode further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

13. The enzyme electrode of claim 7, wherein the enzyme electrode is incorporated into a biosensor configured for subcutaneous continuous glucose measurement, wherein the biosensor is configured to continually measure the amount of glucose in a subject.

14. The enzyme electrode of claim 13, wherein the biosensor comprises the mutated FAD-GDH immobilized onto at least one enzyme electrode, wherein the mutated FAD-GDH is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current, wherein the intensity of the electrical current is indicative of the level of glucose in the subject.

15. The enzyme electrode of claim 14, wherein the enzyme electrode is configured to perform a continuous measurement for up to two weeks.

16. The enzyme electrode of claim 10, wherein the conductive matrix is a conductive polymer, and wherein the conductive polymer is selected from the group consisting of poly(3,4-ethylenedioxythiophene) and Polypyrrol.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.

(2) FIGS. 1A and 1B show some aspects of some embodiments of the present invention. FIG. 1B shows the biochemical response of FAD-GDH F406L to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 1B shows the biochemical response of F406L to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained.

(3) FIG. 2 shows some aspects of an embodiment of the composition of the present invention, showing the electrochemical data in connection with FAD-GDH F406L.

(4) FIGS. 3A-3C show some aspects of some embodiments of the present invention. FIG. 3A shows the biochemical response of FAD-GDH F406A to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 3B shows the biochemical response of F406A to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406A enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 3C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(5) FIGS. 4A-C show some aspects of some embodiments of the present invention. FIG. 4A shows the biochemical response of FAD-GDH F406C to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 4B shows the iochemical response of F406C to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406C enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 4C shows the electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(6) FIGS. 5A-C shows aspects of some embodiments of the present invention. FIG. 5A shows the biochemical response of FAD-GDH F406E to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 5B shows the biochemical response of F406E to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406E enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 5C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(7) FIGS. 6A-C show some aspects of some embodiments of the present invention. FIG. 6A shows the biochemical response of FAD-GDH F406D to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 6B shows the biochemical response of F406D to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406D enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 6C shows the electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(8) FIGS. 7A-C show some aspects of some embodiments of the present invention. FIG. 7A shows the biochemical response of FAD-GDH F406G to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 7B shows the biochemical response of F406G to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406G enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 7C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(9) FIGS. 8A and 8B show aspects of some embodiments of the present invention. FIG. 8A shows the biochemical response of FAD-GDH F406H to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 8B shows the biochemical response of F406H to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained.

(10) FIGS. 9A-C show some aspects of some embodiments of the present invention. FIG. 9A shows the biochemical response of FAD-GDH F406I to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 9B shows the biochemical response of F406I to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406I enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 9C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(11) FIGS. 10A and 10B show some aspects of some embodiments of the present invention. FIG. 10A shows the biochemical response of FAD-GDH F406M to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 10B shows the biochemical response of F406M to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406M enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(12) FIGS. 11A-C show some aspects of some embodiments of the present invention. FIG. 11A shows the biochemical response of FAD-GDH F406N to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 11B shows the biochemical response of F406N to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406N enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 11C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(13) FIGS. 12A and 12B show some aspects of some embodiments of the present invention. FIG. 12A shows the biochemical response of FAD-GDH F406Q to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 12B shows the biochemical response of F406Q to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained.

(14) FIGS. 13A-C show some aspects of some embodiments of the present invention. FIG. 13A shows biochemical response of FAD-GDH F406S to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 13B shows biochemical response of F406S to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406S enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 13C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(15) FIGS. 14A-C show some aspects of some embodiments of the present invention. FIG. 14A shows the biochemical response of FAD-GDH] F406T to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 14B shows biochemical response of F406T to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406T enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 14C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(16) FIGS. 15A-B show negative results. FIG. 15A shows a biochemical response of FAD-GDH F406W to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 15B shows a biochemical response of F406W to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained.

(17) FIGS. 16A-C show some aspects of some embodiments of the present invention. FIG. 16A shows the biochemical response of FAD-GDH F406Y to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 16B shows the biochemical response of F406Y to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406Y enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 16C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(18) FIGS. 17A-C show some aspects of some embodiments of the present invention. FIG. 17A shows the biochemical response of FAD-GDH F406V to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 17B shows the biochemical response of F406V to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406V enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 17C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(19) FIGS. 18A-C show some aspects of some embodiments of the present invention. FIG. 18A shows the biochemical response of FAD-GDH F406P to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 18B shows the biochemical response of F406P to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406P enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 18C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(20) FIGS. 19A-B show some aspects of some embodiments of the present invention. FIG. 19A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215G to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 19B shows the non-linear fit of the data shown in FIG. 19A, from which K.sub.m (k) and V.sub.max have been calculated.

(21) FIGS. 20A-B show some aspects of some embodiments of the present invention. FIG. 20A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215H to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 20B shows the non-linear fit of the data shown in FIG. 20A, from which K.sub.m (k) and V.sub.max have been calculated.

(22) FIGS. 21A-B show some aspects of some embodiments of the present invention. FIG. 21A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215T to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 21B shows the non-linear fit of the data shown in FIG. 21A, from which K.sub.m (k) and V.sub.max have been calculated.

(23) FIGS. 22A-B show some aspects of some embodiments of the present invention. FIG. 22A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215D to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 22B shows the non-linear fit of the data shown in FIG. 22A, from which K.sub.m (k) and V.sub.max have been calculated.

(24) FIGS. 23A-B show some aspects of some embodiments of the present invention. FIG. 23A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215Y to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 23B shows the non-linear fit of the data shown in FIG. 23A, from which K.sub.m (k) and V.sub.max have been calculated.

(25) FIGS. 24A-B show some aspects of some embodiments of the present invention. FIG. 24A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215S to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 24B shows the non-linear fit of the data shown in FIG. 24A, from which K.sub.m (k) and V.sub.max have been calculated.

(26) FIG. 25A shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus), maltose concentrations (shown as a triangle) and xylose (shown as a rectangle) concentrations (substrate solution were supplemented with 1 mM benzoquinone). R.sup.2 represents a linear fit of the glucose data. FIG. 25B shows a biosensor response to glucose and the non-linear fit through which apparent K.sub.m (k) and I.sub.max have been calculated based on Michaelis-Menten

(27) $\begin{array}{cc}I=\frac{I\max \left[S\right]}{\mathrm{Km}+\left[S\right]}& \mathrm{Equation}1\end{array}$
Wherein I is the current, S is the substrate concentration, I.sub.max is the maximum current and the K.sub.m is the apparent Michaelis constant.

(28) FIG. 26A shows an exemplary embodiment of a bioelectrochemical response of an FAD-GDH mutant (N177S, N215S, F353L, F406L) of the present invention, varying concentrations of glucose (shown as a rhombus). FIG. 26B shows a bioelectrochemical response of an FAD-GDH mutant (N177S, N215S, F353L, F406L) of the present invention to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. The enzyme activity of FAD-GDH (N177S, N215S, F353L, F406L) was determined by immobilizing FAD-GDH (N177S, N215S, F353L, F406L) to a carbon electrode by an electropolymerization method without the addition of an electron mediator.

(29) FIG. 27A shows an exemplary embodiment of the present invention, showing a biochemical response of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), maltose (shown as a triangle), an xylose (shown as a rectangle). FIG. 27B shows the biochemical response of FAD-GDH (N177S, N215S, F353L, F406L) to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. FAD-GDH (N177S, N215S, F353L, F406L) activity was determined by monitoring a decrease of dichlorophenolindophenol (DCIP) signal at OD.sub.600 (Epoch Microplate Spectrophotometer, Biotek).

(30) FIGS. 28A and 28B, show an electrochemical response to various substrates. In an exemplary embodiment, FIG. 28A shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus), maltose concentrations (shown as a triangle) and xylose (shown as a rectangle) concentrations. FIG. 28B shows a biosensor response to glucose and the non-linear fit through which apparent K.sub.m (k) and V.sub.max have been calculated. In an exemplary embodiment, wild type FAD-GDH was immobilized to a carbon electrode via an electropolymerization method as described previously. R.sup.2 represents a linear fit of the glucose data. FIG. 28B shows K.sub.mapp=0.85 mM and I.sub.max=1218.5 nA.

(31) FIG. 29A shows biochemical responses of wild type FAD-GDH (w.t.), varying concentrations of glucose (rhombus), maltose (triangle) and xylose (rectangle).

(32) FIG. 29B shows a biochemical response of wild type FAD-GDH to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. The enzyme activity of wild type FAD-GDH was determined by monitoring the decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(33) FIG. 30 is a table showing the results obtained from the biochemical and electrochemical experiments characterizing FAD-GDH (N177S, N215S, F353L, F406L) where: K.sub.m is the Michaelis constant, K.sub.cat is the enzyme's catalytic constant, K.sub.cat/K.sub.m is the catalytic efficiency, glucose/xylose is the ratio of K.sub.catglu/K.sub.catxyl, BEC linearity is the R.sup.2 of the linear fit of the bioelectrochemical experiment, I.sub.20 is the current flux (nA/cm.sup.2) measured when 20 mM of glucose was tested, Xylose selectivity is the ratio of I.sub.20glu/I.sub.20xyl, Maltose selectivity is the ratio of I.sub.20glu/I.sub.20mal, pos1-4, the mutated amino acid number.

(34) FIG. 31 shows sequence data of several mutated FAD-GDH proteins Burkholderia cepacia, SEQ ID NO.: 38; Herbaspirillum seropedicae, SEQ ID NO.: 130 Burkholderia terrae, SEQ ID NO.: 131; Pseudomonas dentrificans, SEQ ID NO.: 132; Yersinia mollaretii, SEQ ID NO.: 133, Rlastonia Pickettii, SEQ ID NO.: 134; Pandoraea sp., SEQ ID NO.: 135) according to some embodiments of the present invention.

(35) FIG. 32 shows a table of electrochemistry data of the embodiments of the composition of the present invention. Mutations in position 406 provide improved linearity over the entire range of physiological range: F406-S/C/T/V/Y/N/P/L/G/A/I/D/E.

(36) FIG. 33 shows some aspects of some embodiments of the present invention. Mutations in position 406 that provide improved selectivity of glucose: F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406W provides an example of a substitution that reduces the enzyme selectivity towards glucose.

(37) FIG. 34A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention. FIG. 34B shows the non-linear fit (red line) of the data represented in FIG. 34A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(38) FIG. 35A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, via direct electron transfer. FIG. 35B shows the non-linear fit (red line) of the data represented in FIG. 35A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(39) FIG. 36A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, via direct electron transfer. FIG. 36B shows the non-linear fit (red line) of the data represented in FIG. 36A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(40) FIG. 37A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, via direct electron transfer. FIG. 37B shows the non-linear fit (red line) of the data represented in FIG. 37A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(41) FIGS. 38A and 38B shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs (FIG. 38A), or 64 hr (FIG. 38B).

(42) FIG. 39 shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs.

(43) FIG. 40 shows a table of electrochemistry data of the embodiments of the electrodes of the present invention.

(44) FIGS. 41A to 41C shows a sequence alignment for multiple different FAD-GDH proteins (SEQ ID NOs: 38, 136-153). For the consensus sequence, uppercase indicates identity, lowercase indicates consensus level of greaters than 0.5, ! is any one of I or V, $ is an one of L or M, % is any one of F or Y, # is any one of NDQEBZ.

(45) Among those benefits and improvements that have been disclosed, other objects and advantages of this invention will become apparent from the following description taken in conjunction with the accompanying figures. Detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely illustrative of the invention that may be embodied in various forms. In addition, each of the examples given in connection with the various embodiments of the invention which are intended to be illustrative, and not restrictive.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

(46) The present invention will be further explained with reference to the attached drawings, wherein like structures are referred to by like numerals throughout the several views. The drawings shown are not necessarily to scale, with emphasis instead generally being placed upon illustrating the principles of the present invention. Further, some features may be exaggerated to show details of particular components.

(47) The figures constitute a part of this specification and include illustrative embodiments of the present invention and illustrate various objects and features thereof. Further, the figures are not necessarily to scale, some features may be exaggerated to show details of particular components. In addition, any measurements, specifications and the like shown in the figures are intended to be illustrative, and not restrictive. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a representative basis for teaching one skilled in the art to variously employ the present invention.

(48) Throughout the specification and claims, the following terms take the meanings explicitly associated herein, unless the context clearly dictates otherwise. The phrases in one embodiment and in some embodiments as used herein do not necessarily refer to the same embodiment(s), though it may. Furthermore, the phrases in another embodiment and in some other embodiments as used herein do not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

(49) In addition, throughout the specification, the meaning of a, an, and the include plural references. The meaning of in includes in and on.

(50) As used herein, linearity refers to the R.sup.2 value of a linear fit which is calculated for the plot of the relation between substrate concentration and the measured current. In some embodiments, good linearity is considered as an R.sup.2 value equal or above 0.85 across the entire range of physiological glucose level (0-600 mg/dL). In some embodiments, good linearity is considered to be an R.sup.2 value that is equal or above 0.9 across the entire range of physiological glucose level. In some embodiments, good linearity is considered as an R.sup.2 value equal or above 0.95 across the entire range of physiological glucose level. Glucose sensing devices transform the measured current to glucose level using a linear based algorithm. Thus the linear range of the glucose sensing enzyme improves the accuracy of measurement of the blood glucose concentration.

(51) As used herein, direct electron transfer means the ability of an enzyme to conduct electrons from its enzymatic core to an electrode, during catalysis, generating a detectable current.

(52) The flavoprotein Glucose dehydrogenase (FAD-GDH, EC 1.1.5.9) is quite recently utilized as a glucose sensing enzyme in glucose test strips. The FAD-GDH catalyzes the oxidation of glucose through the use of various electron acceptors (such as Dichlorophenolindophenol).

(53) To date FAD-GDH has been isolated from gram negative bacteria (Burkholderia cepacia), fungi (Aspergillus sp., A. oryzae, A. niger, A. terreus) and from insects (Drosophila melanogaster, Anopheles gambiae, Apis mellifera, Tribolium castaneum).

(54) The protein is composed of three subunits: a catalytic subunit harboring FAD at its redox center (alpha, 67 kDa), a multiheme electron-transfer subunit (beta, 43 kDa) and a chaperon subunit (gamma, 20 kDa). The alpha subunit can be recombinantly expressed and purified in E. Coli independently of the other subunits, as well as with the beta and the gamma subunits while maintaining catalytic activity in either cases. FAD-GDH is an enzyme that catalyses the oxidation of glucose in the presence of an electron acceptor, such as 2,6-dichlorophenolindophenol or potassium ferricyanide. FAD-GDH can be used in analyte detection assays. FAD-GDH is comprised of multiple subunits, including the catalytic subunit alpha.

(55) In some embodiments the FAD-GDH of the present invention, including all mutants described in the present invention, can be expressed with a beta () subunit (a cytochrome domain) in tandem or on a different plasmid, expressed and purified to generate a protein which can deliver improved electron transfer to various biosensor applications. In some embodiments, the FAD-GDH of the present invention can be expressed and purified without a beta () subunit, as further detailed below.

(56) In some embodiments, analyte detection assays, e.g., glucose detection assays, can be based on the production of hydrogen peroxide and the subsequent detection thereof. For example, glucose is quantitated using assays by first oxidizing glucose with glucose oxidase to produce gluconic acid and hydrogen peroxide. The resultant hydrogen peroxide, in conjunction with a peroxidase, causes the conversion of one or more organic substrates, i.e. an indicator, into a chromogenic product, which product is then detected and related to the glucose concentration in the initial sample.

(57) In the present invention, the inventors have mutated the alpha subunit and co-expressed it with a native gamma subunit. The artificially mutated FAD-GDH alpha (FAD-GDHa) subunit of the present invention can be co-expressed with either a native or a mutated gamma subunit, beta subunit, or both. For example, in some embodiments, the enzyme electrode, configured to measure the amount of glucose in a physiological fluid, comprising the mutated FAD-GDH protein according to some embodiments of the present invention immobilized onto the electrode further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(58) In some embodiments, the present invention is a mutated FAD-GDH protein, wherein the mutated FAD-GDH protein is mutated from a wild-type first species to contain at least one point mutation, wherein the mutated FAD-GDH protein comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of X, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

(59) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

(60) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is X; and d) X.sup.5 is X.

(61) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is X; c) X.sup.4 is X; and d) X.sup.5 is X.

(62) In some embodiments, a) X.sup.1 is X; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is X; and d) X.sup.5 is X.

(63) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is S; and d) X.sup.5 is X.

(64) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is X; and d) X.sup.5 is L.

(65) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is X; c) X.sup.4 is S; and d) X.sup.5 is L.

(66) In some embodiments, a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is S; and d) X.sup.5 is L.

(67) In some embodiments, the amino acid sequence of the mutated FAD-GDH protein comprises the amino acid sequence set forth in any one of SEQ ID NOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.

(68) In some embodiments, a biochemical activity is increased at least 10% compared to a non-mutated FAD-GDH protein from the wild type first species.

(69) In some embodiments, a selectivity for glucose is increased at least 10% compared to a non-mutated FAD-GDH protein from the wild type first species.

(70) In some embodiments, a linearity of current as a function of glucose concentration is increased at least 10% compared to a non-mutated FAD-GDH protein from the wild type first species.

(71) In some embodiments, the FAD-GDH protein has at least one point mutation, wherein the point mutation can be at any one of the amino acid residues selected from the group consisting of: amino acid 177, amino acid 215, amino acid 353, and amino acid 406.

(72) In some embodiments, the FAD-GDH protein has a point mutation at amino acid 406, and at least one additional point mutation, wherein the at least one additional point mutation can be at any one of the amino acid residues selected from the group consisting of: amino acid 177, amino acid 215, and amino acid 353.

(73) In some embodiments, the FAD-GDH protein has point mutations at amino acids 177, 215, and 406.

(74) In some embodiments, the FAD-GDH protein has point mutations at amino acids 215, 353, and 406.

(75) In some embodiments, the FAD-GDH protein has point mutations at amino acids 177, 215, 353, and 406.

(76) A person of ordinary skill in the art would understand that to create a protein with Flavin Adenine DinucleotideGlucose Dehydrogenase alpha subunit (FAD-GDHa) activity according to the instant invention, one would select amino acid residues for such a protein based on the amino acid sequence of a naturally-occurring FAD-GDH protein. The amino acid residues specified in the above sequences (i.e. those residues not identified by X) represent residues that are conserved among FAD-GDH proteins. Such residues serve as a reference point to better specify for the person of ordinary skill in the art the location of amino acid residues in naturally-occurring FAD-GDH (i.e. those identified herein as X.sup.1 and X.sup.2) which can be mutated to create a non naturally-occurring FAD-GDH protein with improved properties.

(77) In some embodiments, the FAD-GDH proteins of the instant invention includes at least one mutation (e.g., a point mutation) in the amino acid sequence, e.g., SEQ ID NOs: 3-8, e.g., as shown in Tables 2 to 6. In some embodiments, the mutation is located at methionine 43 of FAD-GDHa. In some embodiments, the mutation is located at isoleucine 346 of FAD-GDHa. In some embodiments, the mutation is located at serine 420 of FAD-GDHa. In some embodiments, the mutation is located at serine 365 of FAD-GDHa. In some embodiments, the mutation is located at glycine 208 of FAD-GDHa. In some embodiments, the mutation is located at threonine 521 of FAD-GDHa. In some embodiments, the mutation is located at valine 306 of FAD-GDHa. In some embodiments, the mutation is located at glutamine 412 of FAD-GDHa. In some embodiments, the mutation is located at arginine 416 of FAD-GDHa. In some embodiments, the mutation is located at asparagine 215 of FAD-GDHa. In some embodiments, the mutation is located at alanine 487 of FAD-GDHa. In some embodiments, the mutation is located at asparagine 116 of FAD-GDHa. In some embodiments, the mutation is located at asparagine 177 of FAD-GDHa. In some embodiments, the mutation is located at methionine 219 of FAD-GDHa. In some embodiments, the mutation is located at aspartic acid 440 of FAD-GDHa. In some embodiments, the mutation is located at serine 330 of FAD-GDHa. In some embodiments, the mutation is located at proline 257 of FAD-GDHa. In some embodiments, the mutation is located at asparagine 474 of FAD-GDHa. In some embodiments, the mutation is located at threonine 521 of FAD-GDHa. In some embodiments, the mutation is located at serine 420 of FAD-GDHa. In some embodiments, the mutation is located at serine 365 of FAD-GDHa. In some embodiments, the mutation is located at isoleucine 261 of FAD-GDHa. In some embodiments, the mutation is located at threonine 521 of FAD-GDHa. In some embodiments, the mutation is located at proline 173 of FAD-GDHa. In some embodiments, the mutation is located at methionine 219 of FAD-GDHa. In some embodiments, the mutation is located at aspartic acid 301 of FAD-GDHa. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDHa. In some embodiments, the mutation is located threonine 521 of FAD-GDHa. In some embodiments, the mutation is located phenylalanine 406 of FAD-GDHa. In some embodiments, FAD-GDH has at least one mutation (e.g., 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.). In some embodiments, FAD-GDH has at least two mutations (e.g., 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.). In some embodiments, the mutations are point mutations. In some embodiments, asparagine 475 of FAD-GDH is not mutated.

(78) Artificially mutated FAD-GDH protein is meant to refer to a FAD-GDH protein which has at least one amino acid difference from a naturally-occurring FAD-GDH protein. In some embodiments, the present invention is a protein, including: an artificially mutated FAD-GDH protein including at least one mutation, where the at least one mutation (e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.) is at position 406 of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the at least one mutation is selected from the group including: F406S, F406C, F406T, F406M, F406V, F406Y, F406N, F406P, F406L, F406G, F406Q, F406A, F406I, F406D, F406W, F406H, and F406E. In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 10% increase (e.g., but not limited to, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-350% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-300% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-250% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-200% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-150% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-100% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 10%-50% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(79) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(80) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in biochemical activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(81) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 40% increase (e.g., but not limited to, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(82) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 150% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 40% and 100% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(83) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(84) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(85) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 150% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(86) The present invention is a protein, including: an artificially mutated FAD-GDH protein including at least one mutation (e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.), wherein the at least one mutation is at position 474 of SEQ ID NO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutation is selected from the group consisting of: N474H, N474L, N474S and N474V. In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(87) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(88) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(89) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in selectivity compared to a FAD-GDHwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(90) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-150% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(91) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(92) The present invention is a protein, including: an artificially mutated FAD-GDH protein including at least one mutation (e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.), wherein the at least one mutation is at position 177 of SEQ ID NO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutation is N177S.

(93) In some embodiments, the mutation is located at asparagine 177 of FAD-GDH of B. cepacia. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., but not limited to, serine, threonine, cysteine, tyrosine, arginine, and glutamine. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., but not limited to, serine, threonine, and cysteine. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., serine. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to a basic amino acid, e.g., but not limited to, histidine and lysine. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to an acidic amino acid, e.g., but not limited to, aspartic acid and glutamic acid. In some embodiments, the mutation is located at asparagine 177 of FAD-GDH and the asparagine is mutated to a neutral amino acid, e.g., but not limited to, tryptophan, phenylalanine, glycine, alanine, valine, isoleucine, and leucine.

(94) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(95) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(96) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(97) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(98) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-150% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(99) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(100) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(101) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 150% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(102) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(103) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 150% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(104) The present invention is a protein, including: an artificially mutated FAD-GDH protein including at least one mutation (e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.), wherein the at least one mutation is at position 353 of SEQ ID NO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutation is F353L.

(105) In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH of B. cepacia. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the asparagine is mutated to a neutral amino acid, e.g., but not limited to, tryptophan, glycine, alanine, valine, isoleucine, and leucine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to a neutral amino acid, e.g., but not limited to, glycine, alanine, valine, isoleucine, and leucine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to a neutral amino acid, e.g., but not limited to, valine, isoleucine, and leucine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to a neutral amino acid, e.g., but not limited to, leucine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to a basic amino acid, e.g., but not limited to, histidine and lysine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to a polar amino acid, e.g., but not limited to, serine, threonine, cysteine, tyrosine, arginine, asparagine, and glutamine. In some embodiments, the mutation is located at phenylalanine 353 of FAD-GDH and the phenylalanine is mutated to an acidic amino acid, e.g., but not limited to, aspartic acid and glutamic acid.

(106) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(107) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(108) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(109) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(110) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-150% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(111) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(112) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in linearity compared to a FAD-GDHwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(113) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHprotein exhibits between a 20% and 150% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(114) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(115) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 150% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in detectable current via direct electron transport compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(116) The present invention is a protein, including: an artificially mutated FAD-GDH protein including at least one mutation (e.g., but not limited to, 1 mutation, 2 mutations, 3 mutations, 4 mutations, 5 mutations, 6 mutations, 7 mutations, 8 mutations, 9 mutations, 10 mutations, 11 mutations, 12 mutations, 13 mutations, 14 mutations, 15 mutations, etc.), wherein the at least one mutation is at position 215 of SEQ ID NO: 1, or SEQ ID NO: 3. In some embodiments, the at least one mutation is selected from the group consisting of: N215G, N215H, N215T, N215D, N215Y, and N215S.

(117) In some embodiments, the mutation is located at asparagine 215 of FAD-GDH of B. cepacia. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., but not limited to, serine, threonine, cysteine, tyrosine, arginine, and glutamine. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., but not limited to, serine, threonine, and cysteine. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to a polar amino acid, e.g., serine. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to a basic amino acid, e.g., but not limited to, histidine and lysine. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to an acidic amino acid, e.g., but not limited to, aspartic acid and glutamic acid. In some embodiments, the mutation is located at asparagine 215 of FAD-GDH and the asparagine is mutated to a neutral amino acid, e.g., but not limited to, tryptophan, phenylalanine, glycine, alanine, valine, isoleucine, and leucine.

(118) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(119) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(120) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-350% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-300% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in activity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(121) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(122) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-150% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(123) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in selectivity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(124) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially FAD-GDH protein exhibits between a 200% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350% and 500% increase in linearity compared to a FAD-GDHwild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 400% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 450% and 500% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(125) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 200% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDHprotein exhibits between a 20% and 150% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 100% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20% and 50% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50% and 450% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100% and 400% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150% and 350% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200% and 250% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250% and 300% increase in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(126) In some embodiments, the artificially mutated FAD-GDH protein exhibits at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 250%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 300%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 350%-400% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(127) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-350% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-300% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-250% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-200% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-150% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-100% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 20%-50% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(128) In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 50%-350% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 100%-300% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-250% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 200%-250% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, the artificially mutated FAD-GDH protein exhibits between a 150%-200% increase in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(129) In some embodiments, the at least one mutation is selected from the group including: F406S, F406C, F406T, F406M, F406V, F406Y, F406N, F406P, F406L, F406G, F406Q, F406A, F406I, F406D, F406W, F406H, and F406E results in at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in linearity compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39, but a decrease in the current response, compared to a FAD-GDH wild-type protein comprising an amino acid sequence of SEQ ID NO: 38 or SEQ ID NO: 39.

(130) In some embodiments, the at least one mutation is selected from the group consisting of: N215G, N215H, N215T, N215D, N215Y, and N215S results in at least a 20% increase (e.g., but not limited to, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, etc.) in current response compared to a FAD-GDH wild-type protein comprising an amino acid sequence having at least one mutation is selected from the group including: F406S, F406C, F406T, F406M, F406V, F406Y, F406N, F406P, F406L, F406G, F406Q, F406A, F406I, F406D, F406W, F406H, and F406E.

(131) In some embodiments, the protein of the present invention includes at least one mutation at position 406 of SEQ ID NO: 38 or SEQ ID NO: 39, where a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(132) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 95% sequence identity (e.g., but not limited to, 95%, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(133) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 96% sequence identity (e.g., but not limited to, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(134) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 97% sequence identity (e.g., but not limited to, 97%, 97.5%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(135) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 98% sequence identity (e.g., but not limited to, 98%, 98.1%, 98.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(136) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 99% sequence identity (e.g., but not limited to, 99%, 99.1%, 99.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39. In some embodiments, a phenylalanine at the position 406 of SEQ ID NO: 38 or SEQ ID NO: 39 is replaced with any amino acid other than K or R.

(137) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 95% sequence identity (e.g., but not limited to, 95%, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39, where the asparagine residue at position 474 is substituted with valine, histidine, leucine, or serine.

(138) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 96% sequence identity (e.g., but not limited to, 96%, 97%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39, where the asparagine residue at position 474 is substituted with valine, histidine, leucine, or serine.

(139) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 97% sequence identity (e.g., but not limited to, 97%, 97.5%, 98%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39, where the asparagine residue at position 474 is substituted with valine, histidine, leucine, or serine.

(140) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 98% sequence identity (e.g., but not limited to, 98%, 98.1%, 98.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39, where the asparagine residue at position 474 is substituted with valine, histidine, leucine, or serine.

(141) In some embodiments, a mutated FAD-GDH protein, having at least one amino acid mutation, has at least 99% sequence identity (e.g., but not limited to, 99%, 99.1%, 99.2%, etc.) to SEQ ID NO: 38 or SEQ ID NO: 39, where the asparagine residue at position 474 is substituted with valine, histidine, leucine, or serine.

(142) In some embodiments, the present invention is a FAD-GDH protein having at least 95% sequence identity to any of SEQ IDS: 9-29, 40-66, or 86-104. In some embodiments, the present invention is a FAD-GDH protein having at least 96% sequence identity to any of SEQ IDS: 9-29, 40-66, or 86-104. In some embodiments, the present invention is a FAD-GDH protein having at least 97% sequence identity to any of SEQ IDS: 9-29, 40-66, or 86-104. In some embodiments, the present invention is a FAD-GDH protein having at least 98% sequence identity to any of SEQ IDS: 9-29, 40-66, or 86-104. In some embodiments, the present invention is a FAD-GDH protein having at least 99% sequence identity to any of SEQ IDS: 9-29, 40-66, or 86-104.

(143) In some embodiments, the present invention is a FAD-GDH protein of any one of SEQ IDs: 9-29, 40-66, or 86-104 having between 1-5 amino acid mutations. In some embodiments, the present invention is a FAD-GDH protein of any one of SEQ IDs: 9-29, 40-66, or 86-104 having between 1-10 amino acid mutations. In some embodiments, the present invention is a FAD-GDH protein of any one of SEQ IDs: 9-29, 40-66, or 86-104 having between 1-15 amino acid mutations. Proteins of the instant invention are understood to comprise the full length of the amino acid sequence described in such SEQ ID NOs and not a subset.

(144) In some embodiments, the present invention is a FAD-GDH protein (SEQ ID NO: 38 or SEQ ID NO: 39) wherein the FAD-GDH protein includes between 1-15 amino acid substitutions, where at least one amino acid substitution includes a substitution of the phenylalanine at position 406 to another other amino acid other than lysine or arginine.

(145) In some embodiments, the present invention is a FAD-GDH protein (SEQID:1) wherein the FAD-GDH protein includes between 1-15 amino acid substitutions, where at least one amino acid substitution includes a substitution of the arginine at position 474 to a histidine, leucine, serine, or valine.

(146) In some embodiments, the present invention is a method for measuring the glucose level of a subject, the method including: contacting a body fluid obtained from the subject with a mutant FAD-GDH protein, measuring the current generated by the mutant FAD-GDH protein, calculating the measured current to a glucose level, or any combination thereof. In some embodiments, the mutant FAD-GDH protein includes between 1-15 amino acid substitutions including, e.g., but not limited to, a position of 406, 215, or 474 of SEQ ID NO: 38 or SEQ ID NO: 39.

(147) Amino acids of the present invention include, but are not limited to the 20 commonly occurring amino acids. Also included are naturally occurring and synthetic derivatives, for example, selenocysteine. Amino acids further include amino acid analogs. An amino acid analog is a chemically related form of the amino acid having a different configuration, for example, an isomer, or a D-configuration rather than an L-configuration, or an organic molecule with the approximate size and shape of the amino acid, or an amino acid with modification to the atoms that are involved in the peptide bond, so as to be protease resistant when polymerized in a polypeptide.

(148) The phrases amino acid and amino acid sequence as defined here and in the claims can include one or more components which are amino acid derivatives and/or amino acid analogs comprising part or the entirety of the residues for any one or more of the 20 naturally occurring amino acids indicated by that sequence. For example, in an amino acid sequence having one or more tyrosine residues, a portion of one or more of those residues can be substituted with homotyrosine.

(149) The one letter and three letter amino acid codes (and the amino acid that each represents) are as follows: A means ala (alanine); C means cys (cysteine); D means asp (aspartic acid); E means glu (glutamic acid); F means phe (phenylalanine); G means gly (glycine); H means his (histidine); I means ile (isoleucine); K means lys (lysine); L means leu (leucine); M means met (methionine); N means asn (asparagine); P means pro (proline); Q means gln (glutamine); R means arg (arginine); S means ser (serine); T means thr (threonine); V means val (valine); W means trp (tryptophan); and Y means tyr (tyrosine).

(150) Naturally occurring residues are divided into groups based on common side-chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence chain orientation: gly, pro; (6) aromatic: trp, tyr, phe. A person skilled in the art would understand that certain conservative substitution may be made in the amino acid sequence of a protein. Conservative substitutions of interest are shown in Table 1.

(151) TABLE-US-00001 TABLE 1 Exemplary and Preferred Amino acid substitutions. Original Exemplary Preferred Ala (A) val; leu; ile val Arg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) Glu glu Cys (C) Ser ser Gln (Q) Asn asn Glu (E) Asp asp Gly (G) pro; ala ala His (H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe; norleucine leu Leu (L) norleucine; ile; val; met; ala; phe ile Lys (K) arg; gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyr leu Pro (P) Ala ala Ser (S) Thr thr Thr (T) Ser ser Trp (W) tyr; phe tyr Tyr (Y) trp; phe; thr; ser phe Val (V) ile; leu; met; phe; ala; norleucine leu

(152) In some embodiments, the composition of the present invention is directed to a FAD-GDH including at least one point mutation attached to an enzyme electrode configured to: i) recognize a presence of a test subject (for example, glucose); ii) catalyze a redox reaction; and iii) transfer an electron generated from the redox reaction to the enzyme electrode. For purposes of the present invention, catalyze and catalysis refers to activity of specialized proteins (natural or recombinant), called enzymes, which lower the activation energy necessary for a reaction to occur.

(153) In some embodiments, an enzyme electrode is constructed by immobilizing a FAD-GDH alone or FAD-GDH in complex with the gamma subunit on an electrode and/or an electrode layer. In some embodiments, a FAD-GDH is immobilized by utilizing an immobilization method. In some embodiments of the present invention, the immobilization method is selected from the group consisting of: i) using a crosslinking reagent; ii) electropylimerization iii) entrapping an enzyme in a macromolecular matrix; iv) coating the enzyme with a dialysis membrane; and v) immobilizing the enzyme in a polymer, the polymer selected from the group consisting of: 1) a photo-crosslinking polymer; 2) an electric conductive polymer; and 3) a redox polymer. In some embodiments, combinations of the immobilization methods can be utilized.

(154) In some embodiments, the present invention is directed to an enzyme electrode comprising a conductive material and an immobilized or wired FAD-GDH. In some embodiments, the enzyme electrode generates a detectable electric current that corresponds to an amount of an analyte in a tested sample. In some embodiments, the enzyme electrode is an enzyme electrode including an immobilized enzyme on the surface of the enzyme electrode. In some embodiments of the present invention, the enzyme electrode is selected from the group consisting of a gold electrode, a platinum electrode, a carbon electrode, and any other conductive material that is suitable for constructing biosensors. In some embodiments of the present invention, the enzyme electrode utilizes a reaction specificity of an enzyme for detecting any of a variety of biologically active substances in a specific manner.

(155) In some embodiments, the present invention is directed to a sensor including an enzyme electrode to function as a working electrode. In some embodiments of the present invention, the sensor is an assay system for electrochemically measuring a concentration of a test substance. In some embodiments of the present invention, the sensor includes three electrodes: i) a working electrode ii) a counter electrode; and iii) a reference electrode. In some embodiments of the present invention, the sensor comprises a two-electrode system comprising: i) a working electrode and ii) a counter electrode. In some embodiments, the sensor further includes a power source. In some embodiments, the power source applies voltage to: i) the working electrode; ii) an ampere meter; and iii) a recorder. In some embodiments, the sensor is a batch type sensor. In some embodiments, the sensor is a flow type sensor. In some embodiments, the sensor is a flow-type sensor, where the flow-type sensor continuously measures blood glucose level. In some embodiments, the two-electrode system comprising the immobilized enzyme is inserted into a flow of continuously supplied blood sample or dialyzed sample, or into a blood sample or an interstitial fluid sample. In some embodiments, the three-electrode system comprising the immobilized enzyme is inserted into a flow of continuously supplied blood sample or dialyzed sample, or into a blood sample or an interstitial fluid sample.

(156) In some embodiments, the present invention also provides a glucose sensor comprising any of the FAD-GDH proteins of the instant invention. Glucose sensors employing FAD-GDH are known in the art and have been described, for example, in U.S. Pat. No. 8,658,011, which is hereby incorporated by reference. The proteins of the instant invention may be employed in any biosensor know in the art that is designed to employ FAD-GDH.

(157) In some embodiments, the present invention also provides a composition comprising the FAD-GDH of the instant invention and a solid support. Such support may be in the form of a reagent layer or a reagent test strip. In some embodiments the composition is in a dry or solid state. Reagent layers for glucose sensors are known in the art, and have been described, for example, in U.S. Pat. No. 8,658,011. A person skilled in the art would understand that any reagent layer known in the art designed for use with FAD-GDH can be made to comprise the FAD-GDH proteins of the instant invention. Reagent test strips are used in the determination of the concentration of an analyte, e.g. glucose, in a physiological sample, e.g. blood. The test strips can include a porous matrix, one or more members of an analyte oxidation signal producing system and at least one hemolyzing agent. In using the subject test strips for analyte concentration determination, a physiological sample is applied to the test strip. Next, the appearance of a chromogenic product of the signal producing system is detected and related to the concentration of the analyte in the sample.

(158) Typically, a user inserts a test strip into a meter and lances a finger or alternate body site to obtain a blood sample. The drawn sample is applied to the test strip and the meter reads the strip and determines analyte concentration, which is then conveyed to the user. For example, the blood glucose meter converts a current generated by the enzymatic reaction in the test strip to a corresponding blood glucose value which is displayed or otherwise provided to the patient to show the level of glucose at the time of testing.

(159) In some embodiments, the present invention also provides the FAD-GDH proteins of the instant invention immobilized to the conductive component of an electrode of a glucose sensor. Electrodes for glucose sensors are known in the art, and have been described, for example, in U.S. Pat. No. 7,497,940, the contents of which are hereby incorporated by reference. A person skilled in the art would understand that any electrode known in the art designed for use with FAD-GDH can be made to comprise the FAD-GDH proteins of the instant invention.

(160) In some embodiments, the present invention provides an enzyme electrode, configured to measure the amount of glucose in a physiological fluid, comprising the mutated FAD-GDH protein according to some embodiments of the present invention immobilized onto the electrode, wherein the mutated FAD-GDH protein according to some embodiments of the present invention is configured to catalyze glucose in the physiological fluid and produce electrons that are transferred to the electrode thereby generating an electrical current, wherein the intensity of the electrical current is indicative of the level of glucose in the physiological fluid.

(161) As used herein, the term physiological fluid refers to blood, saliva, interstitial fluid, cell culture medium, and the like.

(162) In some embodiments, the enzyme electrode is a screen printed electrode

(163) In some embodiments, the enzyme electrode is configured to perform a single measurement.

(164) An example of an enzyme electrode into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 4,431,507.

(165) Another example of an enzyme electrode into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 5,762,770.

(166) Another example of an enzyme electrode into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 6,270,637.

(167) In some embodiments, the enzyme electrode is incorporated into a glucose test strip.

(168) In some embodiments, the mutated FAD-GDH protein is immobilized on the electrode in a conductive matrix.

(169) In some embodiments, the conductive matrix is selected from a group consisting of carbon paste, graphite paste, graphene oxide, or any other conductive matrix paste appropriate for use in screen printed electrodes.

(170) In some embodiments, the conductive matrix is a conductive polymer.

(171) In some embodiments, the conductive polymer is selected from the group consisting of: PEDOT, and Polypyrrol.

(172) In some embodiments, the conductive polymer is electro-polymerized on the electrode together with the mutated FAD-GDH protein.

(173) In some embodiments, the conductive polymer is chemically polymerized on the electrode together with the mutated FAD-GDH protein.

(174) In some embodiments, the mutated FAD-GDH protein of the present invention is immobilized to the electrode by chemical wiring.

(175) In some embodiments, the conductive matrix further comprises an electron mediator.

(176) In one embodiment, the enzyme electrode, configured to measure the amount of glucose in a physiological fluid, comprising the mutated FAD-GDH protein according to some embodiments of the present invention immobilized onto the electrode further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(177) In some embodiments, the enzyme electrode of the present invention is incorporated into a biosensor configured for subcutaneous continuous glucose measurement, wherein the biosensor is configured to continually measure the amount of glucose in the subject.

(178) In some embodiments, the biosensor is configured to continuously measure glucose for up to 2 weeks. In some embodiments, the biosensor is configured to continuously measure glucose for up to one week. In some embodiments, the biosensor is configured to continuously measure glucose for up to six days. In some embodiments, the biosensor is configured to continuously measure glucose for up to five days. In some embodiments, the biosensor is configured to continuously measure glucose for up to four days. In some embodiments, the biosensor is configured to continuously measure glucose for up to three days. In some embodiments, the biosensor is configured to continuously measure glucose for up to two days. In some embodiments, the biosensor is configured to continuously measure glucose for up to one day.

(179) In some embodiments, the biosensor is configured to continuously measure glucose for up to 64 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 62 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 60 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 58 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 56 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 54 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 52 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 50 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 48 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 46 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 44 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 42 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 40 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 38 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 36 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 34 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 32 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 30 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 28 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 26 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 24 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 20 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 18 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 16 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 14 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 12 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 10 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 8 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 6 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 4 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 2 hours. In some embodiments, the biosensor is configured to continuously measure glucose for up to 1 hour.

(180) An example of a biosensor into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 9,163,273.

(181) Another example of a biosensor into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 8,808,532.

(182) Another example of a biosensor into which a mutated enzyme according to some embodiments of the present invention may be incorporated is disclosed in U.S. Pat. No. 7,074,307.

(183) In some embodiments, the biosensor comprises the mutated FAD-GDH protein according to some embodiments of the present invention immobilized onto at least one enzyme electrode, wherein the mutated FAD-GDH protein according to some embodiments of the present invention is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current, wherein the intensity of the electrical current is indicative of the level of glucose in the subject.

(184) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=6V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of X, S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

(185) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(186) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is selected from the group consisting of S and X; and d) X.sup.5 is selected from the group consisting of L and X.

(187) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDHP subunit, and a wild-type FAD-GDH subunit.

(188) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1 G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X.sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is X; and d) X.sup.5 is X.

(189) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(190) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1 G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X.sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X)=.sub.7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is X; c) X.sup.4 is X; and d) X.sup.5 is X.

(191) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(192) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1 G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X.sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is X; b) X.sup.3 is selected from the group consisting of G, H, D, Y, S, and X; c) X.sup.4 is X; and d) X.sup.5 is X.

(193) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(194) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1 G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X.sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is S; and d) X.sup.5 is X.

(195) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(196) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4 S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, and E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is X; and d) X.sup.5 is L.

(197) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(198) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is X; c) X.sup.4 is S; and d) X.sup.5 is L.

(199) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(200) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises: P(X).sub.n=8X.sup.4(X).sub.n=16V(X).sub.n=6RN(X).sub.n=3YDXRPXCXGX.sup.3NNCMP(X).sub.n=1CP(X).sub.n=2A(X).sub.n=1Y(X).sub.n=1G(X).sub.n=6A(X).sub.n=2AG(X).sub.n=6AVV(X).sub.n=3E(X).sub.n=8-9A(X).sub.n=2Y(X).sub.n=1D(X).sub.n=5HRV(X).sub.n=5V(X).sub.n=2A(X).sub.n=3E(X).sub.n=2K(X).sub.n=4S(X).sub.n=5P(X).sub.n=1G(X).sub.n=2N(X).sub.n=4GRN(X).sub.n=1MDH(X).sub.n=4V(X).sub.n=1F(X).sub.n=6-7W(X).sub.n=1GRGP(X).sub.n=9RDGXX.sup.5R(X).sub.n=19T(X).sub.n=14L(X).sub.n=14X.sup.2(X).sub.n=1X.sup.1(X).sub.n=1E(X).sub.n=4P(X).sub.n=1NR(X).sub.n=3S(X).sub.n=4D(X).sub.n=2G(X).sub.n=7Y(X).sub.n=4Y(X).sub.n=32-35, wherein each X represents a wild-type amino acid residue of the first species and n indicates the number of the wild-type amino acid residues of the first species represented by a respective parenthetical at that position, wherein: a) X.sup.1 is selected from the group consisting of S, C, T, M, V, Y, N, P, L, G, Q, A, I, D, W, H, or E, wherein if X.sup.1 is L, H or V, then X.sup.2 is D; b) X.sup.3 is selected from the group consisting of G, H, D, Y, and S; c) X.sup.4 is S; and d) X.sup.5 is L.

(201) In some embodiments, the mutated FAD-GDH protein further comprises at least one subunit selected from the group consisting of: wild-type FAD-GDH subunit, and a wild-type FAD-GDH subunit.

(202) In some embodiments, the mutated FAD-GDH protein that is configured to catalyze glucose in the subject and generate electrons that are transferred to the electrode and generate electrical current comprises a mutated FAD-GDH having an amino acid sequence set forth in any one of SEQ ID NOS: 9-29, SEQ ID NOS: 46-104, or SEQ ID NOS: 105-129.

(203) In some embodiments, the electrode is made from a material selected from the group consisting of: carbon fiber, graphite, glassy carbon, gold, silver, copper, platinum, palladium, and metal oxide.

(204) In some embodiments, the metal oxide is indium tin oxide.

(205) In some embodiments, mutant FAD-GDH, according to some embodiments of the present invention is immobilized to a carbon electrode via an electropolymerization method. Briefly, the 3-array screen printed electrodes (SPE), which contain working, counter and reference electrodes, is overlaid with 60 l of saturated HKCO.sub.3 solution and voltage applied immediately at 1.2V for 180 seconds (versus Ag/AgCl). The solution is then discarded and washed three times with 60 l of PBS 7.4 supplemented with 0.01M magnesium chloride (EC buffer). The SPE is then overlaid with 60 l of immobilization solution containing of 0.1M pyrrole, 0.1M potassium chloride and 1 mg/ml of SZ2 enzyme. Alternatively, the SPE waiss then overlaid with 60 l of immobilization solution containing of 0.01M PEDOT, 0.7% poly styrene sulfonate (70K Mw) and 1 mg/ml of SZ2 enzyme. An external electron mediator (mediator) can be added to the immobilization solution at the range of 0.5-10 mM. As used herein, electron mediator or mediator refers to a chemical that transfers electrons from the enzyme (e.g., but not limited to, FAD-GDH) to an anode. Electron mediators can include, but are not limited to, Quinone/Hydroquinone, Phenanthroline quinone, Quinone diimine/Phenylendiamine, Qunone diimine oxides (Nitrosoanilines), Phenazinium/-radical/Dihydro-phenazine, N-oxides (Resazurin, Bezfuroxan), Hexacyanoferrate III/III, Ferricinium/Ferrocene (carboxylic acid), Ruthenium complexes, Osmium as complexed and/or immobilized state and any other mediators common in the art. The solution was incubated at room temperature (RT) for 1 minute prior to applying a set of 10 pulses at 0.65V for 1 second with 5 second relaxation. The solution was then discarded and washed three times with 60 l of EC buffer for initiation of glucose sensing measurement phase.

(206) In the embodiments utilizing PEDOT, the solution is incubated at room temperature (RT) for 1 minute prior to applying 2 cyclic voltammetry cycles (0.2-0.9V 50 mv/sec).

(207) In some embodiments, the electrode is overlaid with immobilization solution devoid of any mediator as indicated in the table below. These embodiments enable direct electron transfer (DET). In some embodiments, the immobilization solution is electropolymerized onto the electrode as described in the table below. The solution is then discarded and washed three times with 60 l of EC buffer for initiation of glucose sensing measurements phase as detailed in the table below. The electrochemical data was collected using a VMP3 multi-channel potentiostat (Bio-logic Science instruments SAS, France).

(208) TABLE-US-00002 Composition Polymerization Glucose # description method sensing 1 0.25M pyrrole, 0.25M 20 CA pulses 0.2 V potassium chloride, of 0.65 V, for 30 Phosphate Buffer Saline 1 second each with minutes pH 7.4 supplemented 5 second intervals with 0.01 mM MgCl.sub.2 and 0.1% Proclin 150 (Sigma, cat# 49376-u) (PBSm) and 0.33 mg/ml of Mut111 enzyme between each pulse 2 0.01M 3,4-Ethylenedioxythiophene CV cycles ranging 0.3 V (EDOT), 0.1 mM Ply(sodium 0.2-0.85 V for 30 4-styrenesulfonate) (PSS), with a scanning minutes PBSm and 1 mg/ml speed of of Mut111 enzyme 0.05 V/Sec

(209) Examples of methods by which mutated enzyme according to some embodiments of the present invention may be incorporated into a biosensor are disclosed in U.S. Pat. No. 9,163,273.

(210) Other examples of methods by which mutated enzyme according to some embodiments of the present invention may be incorporated into a biosensor are disclosed in U.S. Pat. No. 8,808,532.

(211) Although the following exemplary embodiments were conducted with non-naturally mutated FAD-GDH derived from B. cepacia, these exemplary embodiments are non-limiting and can be extended to related bacterial FAD-GDH, such as, but not limited to, FAD-GDH derived from B. lata, Burkholderia terrae, Pseudomonas denitrificans, Relsonia pickettii, Yersinia mollretii, Pandoraea sp. and Herbspirilum sropedicae. FIG. 7 illustrates the homology of the following species: B. cepacia, H. Seropedicae, B. terrae, P. Dentrificans, Y. mollaretii, R. Pickettii, and Pandoraea. The positions described herein are noted using ellipsoid shapes on FIG. 31.

(212) The following electrochemical exemplary embodiments use conductive polymers, e.g., polypyrrole. In some embodiments, a conductive polymer is used to generate electrochemical data and includes polypyrrole, polythiophene (e.g., poly(3,4-ethylenedioxythiophene)PEDOT), polyaniline, poly(diphenylamine), polyazine, poly(o-aminophenol, or any combination thereof.

(213) In some embodiments, the present invention also provides a DNA sequence encoding any of the proteins of the instant invention. Such DNA sequence may be expressed according to any technique known in the art. By way of non-limiting example, such sequence may be incorporated into a vector for expression in a cell. The term vector refers to a polynucleotide molecule capable of carrying and transferring another polynucleotide fragment or sequence to which it has been linked from one location (e.g., a host, a system) to another. The term includes vectors for in vivo or in vitro expression systems. As a non-limiting example, vectors can be in the form of plasmids which refer to circular double stranded DNA loops which are typically maintained episomally but may also be integrated into the host genome. In some embodiments, the present invention accordingly provides a host cell comprising the DNA encoding the protein and a process of producing the protein comprising culturing the host cell under conditions conducive to the production of the protein, and recovering the protein.

(214) Exemplary embodiments of the activity of wild type FAD-GDH (SEQ ID NO: 38) is shown in International Application Serial No. PCT/US2015/64125, entitled Compositions and Methods for Measuring Blood Glucose Levels, filed on Dec. 4, 2015.

(215) Exemplary embodiments of the activity of mutated GAD-GDH is in International Application Serial No. PCT/US2015/64125, entitled Compositions and Methods for Measuring Blood Glucose Levels, filed on Dec. 4, 2015.

(216) Exemplary embodiments of the composition of the present invention are shown in FIGS. 1A and 1B. FIG. 1A shows the biochemical response of FAD-GDH F406L to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 1B shows the biochemical response of F406L to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406L enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(217) An exemplary embodiment of the composition of the present invention, showing the electrochemical data in connection with FAD-GDH F406L, is shown in FIG. 2.

(218) Exemplary embodiments of the composition of the present invention are shown in FIGS. 3A-3C. FIG. 3A shows the biochemical response of FAD-GDH F406A to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 3B shows the biochemical response of F406A to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406A enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 3C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(219) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 4A-C. FIG. 4A shows the biochemical response of FAD-GDH F406C to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 4B shows the iochemical response of F406C to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406C enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 4C shows the electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(220) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 5A-C. FIG. 5A shows the biochemical response of FAD-GDH F406E to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 5B shows the biochemical response of F406E to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406E enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 5C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(221) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 6A-C. FIG. 6A shows the biochemical response of FAD-GDH F406D to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 6B shows the biochemical response of F406D to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406D enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 6C shows the electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(222) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 7A-C. FIG. 7A shows the biochemical response of FAD-GDH F406G to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 7B shows the biochemical response of F406G to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406G enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 7C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(223) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 8A and 8B. FIG. 8A shows the biochemical response of FAD-GDH F406H to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 8B shows the biochemical response of F406H to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406H enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(224) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 9A-C. FIG. 9A shows the biochemical response of FAD-GDH F406I to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 9B shows the biochemical response of F406I to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406I enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 9C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(225) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 10A and 10B. FIG. 10A shows the biochemical response of FAD-GDH F406M to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 10B shows the biochemical response of F406M to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406M enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(226) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 11A-C. FIG. 11A shows the biochemical response of FAD-GDH F406N to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 11B shows the biochemical response of F406N to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406N enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 11C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(227) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 12A and 12B. FIG. 12A shows the biochemical response of FAD-GDH F406Q to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 12B shows the biochemical response of F406Q to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406Q enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(228) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 13A-C. FIG. 13A shows biochemical response of FAD-GDH F406S to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 13B shows the biochemical response of F406S to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406S enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 13C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(229) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 14A-C. FIG. 14A shows the biochemical response of FAD-GDH F406T to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 14B shows biochemical response of F406T to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406T enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 14C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(230) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 15A-B. FIG. 15A shows a biochemical response of FAD-GDH F406W to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 15B shows a biochemical response of F406W to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been obtained. F406W enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(231) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 16A-C. FIG. 16A shows the biochemical response of FAD-GDH F406Y to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 16B shows the biochemical response of F406Y to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406Y enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 16C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(232) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 17A-C. FIG. 17A shows the biochemical response of FAD-GDH F406V to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 17B shows the biochemical response of F406V to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406V enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 17C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(233) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 18A-C. FIG. 18A shows the biochemical response of FAD-GDH F406P to varying concentrations of glucose (rhombus), and xylose (rectangle). FIG. 18B shows the biochemical response of F406P to glucose and the non-linear fit through which K.sub.m(k) and V.sub.max have been obtained. F406P enzyme activity was determined via monitoring decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600. FIG. 18C shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus) and the linear fit across the data range is represented by the R.sup.2 value.

(234) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 19A-B. FIG. 19A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215G to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 19B shows the non-linear fit of the data shown in FIG. 19A, from which K.sub.m (k) and V.sub.max have been calculated.

(235) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 20A-B. FIG. 20A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215H to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 20B shows the non-linear fit of the data shown in FIG. 20A, from which K.sub.m (k) and V.sub.max have been calculated.

(236) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 21A-B. FIG. 21A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215T to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 21B shows the non-linear fit of the data shown in FIG. 21A, from which K.sub.m (k) and V.sub.max have been calculated.

(237) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 22A-B. FIG. 22A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215D to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 22B shows the non-linear fit of the data shown in FIG. 22A, from which K.sub.m (k) and V.sub.max have been calculated.

(238) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 23A-B. FIG. 23A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215Y to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 23B shows the non-linear fit of the data shown in FIG. 23A, from which K.sub.m (k) and V.sub.max have been calculated.

(239) Exemplary embodiments of the compositions of the present invention are shown in FIGS. 24A-B. FIG. 24A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215S to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 24B shows the non-linear fit of the data shown in FIG. 24A, from which K.sub.m (k) and V.sub.max have been calculated.

(240) Exemplary embodiments showing electrochemistry data in connection with the wild type FAD-GDH protein are shown in FIG. 25.

(241) Exemplary embodiments are shown in a table listing electrochemistry data of the composition of the present invention in FIG. 26. Mutations in position 406 provide improved linearity over the entire range of physiological range: F406-S/C/T/V/Y/N/P/L/G/A/I/D/E. Mutations in position 215 provide improved linearity over the entire range of physiological range: N215-G/H/T/D/Y/S.

(242) Exemplary embodiments of the composition of the present invention are shown in FIG. 27. Mutations in position 406 that provide improved selectivity of glucose: F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406W provides an example of a substitution that reduces the enzyme selectivity towards glucose.

(243) FIG. 34A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with polypyrrole, and benzoquinone is used as a mediator to obtain a single measurement. FIG. 34B shows the non-linear fit (red line) of the data represented in FIG. 34A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(244) FIG. 35A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with polypyrrole, to obtain a single measurement via direct electron transfer. FIG. 35B shows the non-linear fit (red line) of the data represented in FIG. 35A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(245) FIG. 36A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with PEDOT, to obtain a single measurement via direct electron transfer. FIG. 36B shows the non-linear fit (red line) of the data represented in FIG. 36A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(246) FIG. 37A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with graphene oxide, to obtain a single measurement via direct electron transfer. FIG. 37B shows the non-linear fit (red line) of the data represented in FIG. 37A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(247) FIGS. 38A and 38B shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs (FIG. 38A), or 64 hr (FIG. 38B). In this the mutant FAD-GDH protein is immobilized with polypyrrole, to obtain a single measurement via direct electron transfer.

(248) FIG. 39 shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs. In this embodiment, the mutant FAD-GDH protein is immobilized with PEDOT, to obtain a single measurement via direct electron transfer

(249) FIG. 40 shows a table of electrochemistry data of the embodiments of the electrodes of the present invention.

(250) Exemplary compositions comprising electrodes according to some embodiments of the present invention are shown in FIGS. 39 and 40. Mutations at positions 177, 215, 353, and 406 provide improved linearity, selectivity and detectable current via direct electron transfer over the entire physiological range. Further, the electrodes according to some embodiments of the present invention are capable of measuring glucose for up to 64 hours.

(251) In some embodiments, the protein of the present invention can be linked to an epitope tag, e.g., but not limited to, a HIS tag, a 6HIS tag, a maltose binding protein tag, a green fluorescent protein tag, a glutathione-s-transferase tag, a streptavidin tag, etc. The epitope tag can be separated from the protein by using a linker having, e.g., 1-n amino acids in length.

Examples: Mutated FAD-GDH, Alpha Subunit (), Strain B. cepacia

(252) Examples of mutated FAD-GDH proteins are disclosed in International Application Serial No. PCT/US2015/64125, entitled Compositions and Methods for Measuring Blood Glucose Levels, filed on Dec. 4, 2015.

Example 1

(253) FAD-GDH was mutated to include the following point mutation: N215D as shown in SEQ ID NO: 105.

Example 2

(254) FAD-GDH was mutated to include the following point mutation: N215G as shown in SEQ ID NO: 106.

Example 3

(255) FAD-GDH was mutated to include the following point mutation: N215H as shown in SEQ ID NO: 107.

Example 4

(256) FAD-GDH was mutated to include the following point mutation: N215T as shown in SEQ ID NO: 108.

Example 5

(257) FAD-GDH was mutated to include the following point mutation: N215Y as shown in SEQ ID NO: 109.

Example 6

(258) B. sepacia FAD-GDH was mutated to include the following point mutation: N177S, N215S, F353L, F406L, as shown in SEQ ID NO: 110.

Example 7

(259) B. lata FAD-GDH was mutated to include the following point mutation: N177S, N215S, F353L, F406L, as shown in SEQ ID NO: 111.

Example 8

(260) B. lata FAD-GDH was mutated to include the following point mutation: N215S, as shown in SEQ ID NO: 112.

Example 9

(261) B. lata FAD-GDH was mutated to include the following point mutation: N215T, as shown in SEQ ID NO: 113.

Example 10

(262) B. cepacia FAD-GDH was mutated to include the following point mutation: N177S, as shown in SEQ ID NO: 114.

Example 11

(263) B. lata FAD-GDH was mutated to include the following point mutation: N177S, as shown in SEQ ID NO: 115.

Example 12

(264) B. cepacia FAD-GDH was mutated to include the following point mutation: F353L, as shown in SEQ ID NO: 116.

Example 13

(265) B. lata FAD-GDH was mutated to include the following point mutation: F353L, as shown in SEQ ID NO: 117.

Example 14

(266) B. cepacia FAD-GDH was mutated to include the following point mutation: N177S, F406L, as shown in SEQ ID NO: 118.

Example 15

(267) B. lata FAD-GDH was mutated to include the following point mutation: N177S, F406L, as shown in SEQ ID NO: 119.

Example 16

(268) B. sepacia FAD-GDH was mutated to include the following point mutation: F353L, F406L, as shown in SEQ ID NO: 120.

Example 17

(269) B. lata FAD-GDH was mutated to include the following point mutation: F353L, F406L, as shown in SEQ ID NO: 121.

Example 18

(270) B. sepacia FAD-GDH was mutated to include the following point mutation: N215S, F406L, as shown in SEQ ID NO: 122.

Example 19

(271) B. lata FAD-GDH was mutated to include the following point mutation: N215S, F406L, as shown in SEQ ID NO: 123.

Example 20

(272) B. sepacia FAD-GDH was mutated to include the following point mutation: N177S, F353L, F406L, as shown in SEQ ID NO: 124.

Example 21

(273) B. lata FAD-GDH was mutated to include the following point mutation: N177S, F353L, F406L, as shown in SEQ ID NO: 125.

Example 22

(274) B. sepacia FAD-GDH was mutated to include the following point mutation: N177S, N215S, F406L, as shown in SEQ ID NO: 126.

Example 23

(275) B. lata FAD-GDH was mutated to include the following point mutation: N177S, N215S, F406L, as shown in SEQ ID NO: 127.

Example 24

(276) B. sepacia FAD-GDH was mutated to include the following point mutation: N215S, F353L, F406L, as shown in SEQ ID NO: 128.

Example 25

(277) B. lata FAD-GDH was mutated to include the following point mutation: N215S, F353L, F406L, as shown in SEQ ID NO: 129.

(278) The proteins described in Examples 1-25 were generated according to the methods described in International Application Serial No. PCT/US2015/64125, entitled Compositions and Methods for Measuring Blood Glucose Levels, filed on Dec. 4, 2015.

(279) TABLE-US-00003 TABLE 2 Protein sequences of FAD-GDHfrom B. cepacia (SEQ ID NO: 1, 3-29) and B. lata (SEQ ID NO: 2) and DNA sequences of primers used for random mutagenesis via error prone PCR. Each protein sequence ends in a removable 6x-His tag. SEQ ID Sequence SEQ ID NO: 1 Protein sequence ORF2 (FAD) - B. cepacia Wild type MADTDTQKADVVVVGSGVAGATVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPOPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 2 Protein sequence ORF2 (FAD) - B. lata Wild type MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 3 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWE IVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIR AVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQR AEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLVGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTADAIGIPRPEITYA IDDYVKRGAAHIREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 4 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLFNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPNKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 5 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 6 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVALTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 7 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGAANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPRPENCIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 8 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLTDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSHIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 9 Protein sequence ORF2 (FAD) - B. cepacia F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 10 Protein sequence ORF2 (FAD) - B. cepacia F406D mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCDHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 11 Protein sequence ORF2 (FAD) - B. cepacia F406H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCHHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 12 Protein sequence ORF2 (FAD) - B. cepacia F406M mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCMHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 13 Protein sequence ORF2 (FAD) - B. cepacia F406E mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCEHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 14 Protein sequence ORF2 (FAD) - B. cepacia F406S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCSHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 15 Protein sequence ORF2 (FAD) - B. cepacia F406T mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCTHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 16 Protein sequence ORF2 (FAD) - B. cepacia F406Y mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCYHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 17 Protein sequence ORF2 (FAD) - B. cepacia F406N mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCNHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 18 Protein sequence ORF2 (FAD) - B. cepacia F406Q mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCQHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 19 Protein sequence ORF2 (FAD) - B. cepacia F406C mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCCHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 20 Protein sequence ORF2 (FAD) - B. cepacia F406G mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCGHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 21 Protein sequence ORF2 (FAD) - B. cepacia F406P mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCPHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 22 Protein sequence ORF2 (FAD) - B. cepacia F406A mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCAHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 23 Protein sequence ORF2 (FAD) - B. cepacia F406V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCVHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 24 Protein sequence ORF2 (FAD) - B. cepacia F406I mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCIHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 25 Protein sequence ORF2 (FAD) - B. cepacia F406W mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCWHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEVGSGSGHHHHHH SEQ ID NO: 26 Protein sequence ORF2 (FAD) - B. cepacia N474H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPHNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 27 Protein sequence ORF2 (FAD) - B. cepacia N474L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPLNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 28 Protein sequence ORF2 (FAD) - B. cepacia N474S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPSNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 29 Protein sequence ORF2 (FAD) - B. cepacia N474V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPVNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 30 Primer used for random mutagenesis via error prone PCR method CCAGGCAAATTCTGTTTTATCAGACC SEQ ID NO: 31 Primer used for random mutagenesis via error prone PCR method CAAGCTGGAGACCGTTTAAACTC SEQ ID NO: 32 Primer used for random mutagenesis via error prone PCR method CGCTATTCAGATCCTCTTCTGAGATG SEQ ID NO: 33 Primer used for random mutagenesis via error prone PCR method GCTTCTGCGTTCTGATTTAATCTG SEQ ID NO: 34 Primer used for random mutagenesis via error prone PCR method GGTCGTGGTCGGATCCGGTGTGGCAGGTGCTATTGTG SEQ ID NO: 35 Primer used for random mutagenesis via error prone PCR method CGTTCTTATTGCCCGAATAAACC SEQ ID NO: 36 Primer used for random mutagenesis via error prone PCR method CGAAGAAGCCCTGATGTTTGG SEQ ID NO: 37 Primer used for random mutagenesis via error prone PCR method GAAGCATGGTATCTGGGCATTGTTG Bold text indicates mutations relative to wild-type within the amino acid sequence

(280) TABLE-US-00004 TABLE 3 Protein sequences of FAD-GDHya from B. cepacia (SEQ ID NO: 38, SEQ ID NOs: 40-66) and B. lata (SEQ ID NO: 39). SEQ ID NO: 38 Protein sequence ORF2 (FAD) - B. cepacia Wild type MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 39 Protein sequence ORF2 (FAD) B. lata Wild type MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFFI VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 40 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWE IVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIR AVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQR AEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLVGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 41 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLFNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRTVPNKTATDATGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 42 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 43 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVALTIAALALRMSDT LKKEV SEQ ID NO: 44 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGAANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPRPENCIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 45 Protein sequence ORF2 (FAD) - B. cepacia mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLTDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSHIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 46 Protein sequence ORF2 (FAD) - B. cepacia F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 47 Protein sequence ORF2 (FAD) - B. cepacia F406D mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCDHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 48 Protein sequence ORF2 (FAD) - B. cepacia F406H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCHHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 49 Protein sequence ORF2 (FAD) - B. cepacia F406M mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCMHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 50 Protein sequence ORF2 (FAD) - B. cepacia F406E mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCEHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 51 Protein sequence ORF2 (FAD) - B. cepacia F406S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCSHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 52 Protein sequence ORF2 (FAD) - B. cepacia F406T mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCTHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 53 Protein sequence ORF2 (FAD) - B. cepacia F406Y mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCYHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 54 Protein sequence ORF2 (FAD) - B. cepacia F406N mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCNHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 55 Protein sequence ORF2 (FAD) - B. cepacia F406Q mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCQHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 56 Protein sequence ORF2 (FAD) - B. cepacia F406C mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCCHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 57 Protein sequence ORF2 (FAD) - B. cepacia F406G mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCGHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 58 Protein sequence ORF2 (FAD) - B. cepacia F406P mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCPHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 59 Protein sequence ORF2 (FAD) - B. cepacia F406A mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCAHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 60 Protein sequence ORF2 (FAD) - B. cepacia F406V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCVHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 61 Protein sequence ORF2 (FAD) - B. cepacia F406I mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCIHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 62 Protein sequence ORF2 (FAD) - B. cepacia F406W mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCWHEILPQPENRIVPSKTATDAIGIPRPEITY AIDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMG ADARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSD TLKKEV SEQ ID NO: 63 Protein sequence ORF2 (FAD) - B. cepacia N474H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPHNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 64 Protein sequence ORF2 (FAD) - B. cepacia N474L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPLNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 65 Protein sequence ORF2 (FAD) - B. cepacia N474S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPSNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 66 Protein sequence ORF2 (FAD) - B. cepacia N474V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPVNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEV

(281) TABLE-US-00005 TABLE 4 Protein sequences of FAD-GDHya for B. lata mutants with removable 6x-His tag. SEQ ID Sequence SEQ ID NO: 67 Protein sequence ORF2 (FAD) - B. lata F406A mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCAHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 68 Protein sequence ORF2 (FAD) - B. lata F406C mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCCHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 69 Protein sequence ORF2 (FAD) - B. lata F406D mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCDHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 70 Protein sequence ORF2 (FAD) - B. lata F406E mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCEHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 71 Protein sequence ORF2 (FAD) - B. lata F406G mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCGHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 72 Protein sequence ORF2 (FAD) - B. lata F406H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCHHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 73 Protein sequence ORF2 (FAD) - B. lata F406I mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCIHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 74 Protein sequence ORF2 (FAD) - B. lata F406K mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCKHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 75 Protein sequence ORF2 (FAD) - B. lata F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 76 Protein sequence ORF2 (FAD) - B. lata F406M mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCMHEILPQPENRIVPSKTATDAVGIPRPEITYA IDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 77 Protein sequence ORF2 (FAD) - B. lata F406N mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCNHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 78 Protein sequence ORF2 (FAD) - B. lata F406Q mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCQHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 79 Protein sequence ORF2 (FAD) - B. lata F406S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCSHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 80 Protein sequence ORF2 (FAD) - B. lata F406P mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCPHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 81 Protein sequence ORF2 (FAD) - B. lata F406V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCVHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 82 Protein sequence ORF2 (FAD) - B. lata F406R mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCRHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 83 Protein sequence ORF2 (FAD) - B. lata F406T mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCTHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 84 Protein sequence ORF2 (FAD) - B. lata F406W mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCWHEILPQPENRIVPSKTATDAVGIPRPEITYA IDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH* SEQ ID NO: 85 Protein sequence ORF2 (FAD) - B. lata F406Y mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCYHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH*

(282) TABLE-US-00006 TABLE 5 Protein sequences of FAD-GDHya for B. lata mutants. SEQ ID Sequence SEQ ID NO: 86 Protein sequence ORF2 (FAD) - B. lata F406A mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCAHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 87 Protein sequence ORF2 (FAD) - B. lata F406C mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCCHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 88 Protein sequence ORF2 (FAD) - B. lata F406D mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCDHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 89 Protein sequence ORF2 (FAD) - B. lata F406E mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCEHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 90 Protein sequence ORF2 (FAD) - B. lata F406G mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCGHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 91 Protein sequence ORF2 (FAD) - B. lata F406H mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCHHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 92 Protein sequence ORF2 (FAD) - B. lata F406I mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCIHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 93 Protein sequence ORF2 (FAD) - B. lata F406K mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCKHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 94 Protein sequence ORF2 (FAD) - B. lata F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 95 Protein sequence ORF2 (FAD) - B. lata F406M mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCMHEILPQPENRIVPSKTATDAVGIPRPEITYA IDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 96 Protein sequence ORF2 (FAD) - B. lata F406N mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCNHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 97 Protein sequence ORF2 (FAD) - B. lata F406Q mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCQHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 98 Protein sequence ORF2 (FAD) - B. lata F406S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCSHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 99 Protein sequence ORF2 (FAD) - B. lata F406P mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCPHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEV SEQ ID NO: 100 Protein sequence ORF2 (FAD) - B. lata F406V mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCVHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO: 101 Protein sequence ORF2 (FAD) - B. lata F406R mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCRHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO: 102 Protein sequence ORF2 (FAD) - B. lata F406T mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCTHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO: 103 Protein sequence ORF2 (FAD) - B. lata F406W mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCWHEILPQPENRIVPSKTATDAVGIPRPEITYA IDDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG SEQ ID NO: 104 Protein sequence ORF2 (FAD) - B. lata F406Y mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCYHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSG

(283) TABLE-US-00007 TABLE 6 Protein sequences according to some embodiments of the present invention. SEQ ID Sequence SEQ ID NO: 105 Protein sequence ORF2 (FAD) - N215D MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGDNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 106 Protein sequence ORF2 (FAD) - N215G MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGGNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 107 Protein sequence ORF2 (FAD) - N215H MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGHNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 108 Protein sequence ORF2 (FAD) - N215T MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGTNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO. 109 Protein sequence ORF2 (FAD) - N215Y MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGYNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 110 Protein sequence ORF2 (FAD) - B. cepacia N177S, N215S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRTPRWE IVERFRNQPDKMDFMAPYPSSWAPHPEYGPPNDYLILKGEHKFNSQYIR AVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQR AEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFSEQTIKTALNNYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAERAG AKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGIE TPRILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPGR GPQEMTSLVGFRDGPLRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNDHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 111 Protein sequence ORF2 (FAD) - B. lata N177S, N215S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFSEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 112 Protein sequence ORF2 (FAD) - B. lata N215S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 113 Protein sequence ORF2 (FAD) - B. lata N215T mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGTNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 114 Protein sequence ORF2 (FAD) - B. cepacia N177S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFSEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 115 Protein sequence ORF2 (FAD) - B. lata N177S mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFSEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 116 Protein sequence ORF2 (FAD) - B. cepacia F353L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPLRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCFHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 117 Protein sequence ORF2 (FAD) - B. lata F353L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCFHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 118 Protein sequence ORF2 (FAD) - B. cepacia N177S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFSEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 119 Protein sequence ORF2 (FAD) - B. lata N177S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFSEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 120 Protein sequence ORF2 (FAD) - B. cepacia F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAER AGAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANG IETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWP GRGPQEMTSLIGFRDGPLRATEAAKKIHLSNLSRIDQETQKIFKAGKLMK PDELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYA IDDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 121 Protein sequence ORF2 (FAD) - B. lata F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 122 Protein sequence ORF2 (FAD) - B. cepacia N215S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 123 Protein sequence ORF2 (FAD) - B. lata N215S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 124 Protein sequence ORF2 (FAD) - B. cepacia N177S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFSEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPLRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 125 Protein sequence ORF2 (FAD) - B. lata N177S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFSEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGNNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 126 Protein sequence ORF2 (FAD) - B. cepacia N177S, N215S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFSEQTIKTALNNYDPKF HVVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPFRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 127 Protein sequence ORF2 (FAD) - B. lata N177S, N215S, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFSEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPFRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 128 Protein sequence ORF2 (FAD) - B. cepacia N215S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKAVILLEAGPRMPRW EIVERFRNQPDKMDFMAPYPSSPWAPHPEYGPPNDYLILKGEHKFNSQY IRAVGGTTWHWAASAWRFIPNDFKMKSVYGVGRDWPIQYDDLEPYYQ RAEEELGVWGPGPEEDLYSPRKQPYPMPPLPLSFNEQTIKTALNNYDPK FHVVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAERA GAKLIENAVVYKLETGPDKRIVAALYKDKTGAEHRVEGKYFVLAANGI ETPKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYASEKLWPG RGPQEMTSLIGFRDGPLRATEAAKKIHLSNLSRIDQETQKIFKAGKLMKP DELDAQIRDRSARYVQFDCLHEILPQPENRIVPSKTATDAIGIPRPEITYAI DDYVKRGAAHTREVYATAAKVLGGTDVVFNDEFAPNNHITGSTIMGA DARDSVVDKDCRTFDHPNLFISSSATMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH SEQ ID NO: 129 Protein sequence ORF2 (FAD) - B. lata N215S, F353L, F406L mutant MADTDTQKADVVVVGSGVAGAIVAHQLAMAGKSVILLEAGPRMPRWE IVERFRNQVDKTDFMAPYPSSAWAPHPEYGPPNDYLILKGEHKFNSQYI RAVGGTTWHWAASAWRFIPNDFKMKTVYGVGRDWPIQYDDIEHYYQR AEEELGVWGPGPEEDLYSPRKEPYPMPPLPLSFNEQTIKSALNGYDPKFH VVTEPVARNSRPYDGRPTCCGSNNCMPICPIGAMYNGIVHVEKAEQAG AKLIDSAVVYKLETGPDKRITAAVYKDKTGADHRVEGKYFVIAANGIET PKILLMSANRDFPNGVANSSDMVGRNLMDHPGTGVSFYANEKLWPGR GPQEMTSLIGFRDGPLRANEAAKKIHLSNMSRINQETQKIFKGGKLMKP EELDAQIRDRSARFVQFDCLHEILPQPENRIVPSKTATDAVGIPRPEITYAI DDYVKRGAVHTREVYATAAKVLGGTEVVFNDEFAPNNHITGATIMGA DARDSVVDKDCRAFDHPNLFISSSSTMPTVGTVNVTLTIAALALRMSDT LKKEVGSGSGHHHHHH

(284) FIGS. 19A-B show some aspects of some embodiments of the present invention. FIG. 19A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215G to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 19B shows the non-linear fit of the data shown in FIG. 19A, from which K.sub.m (k) and V.sub.max have been calculated.

(285) FIGS. 20A-B show some aspects of some embodiments of the present invention. FIG. 20A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215H to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 20B shows the non-linear fit of the data shown in FIG. 20A, from which K.sub.m (k) and V.sub.max have been calculated.

(286) FIGS. 21A-B show some aspects of some embodiments of the present invention. FIG. 21A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215T to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 21B shows the non-linear fit of the data shown in FIG. 21A, from which K.sub.m (k) and V.sub.max have been calculated.

(287) FIGS. 22A-B show some aspects of some embodiments of the present invention. FIG. 22A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215D to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 22B shows the non-linear fit of the data shown in FIG. 22A, from which K.sub.m (k) and V.sub.max have been calculated.

(288) FIGS. 23A-B show some aspects of some embodiments of the present invention. FIG. 23A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215Y to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 23B shows the non-linear fit of the data shown in FIG. 23A, from which K.sub.m (k) and V.sub.max have been calculated.

(289) FIGS. 24A-B show some aspects of some embodiments of the present invention. FIG. 24A shows electrochemical data representing the current response of the biosensor comprising of FAD-GDH N215S to various glucose concentrations (shown as a square) and the linear fit across the data range is represented by the R.sup.2 value. FIG. 24B shows the non-linear fit of the data shown in FIG. 24A, from which K.sub.m (k) and V.sub.max have been calculated.

(290) FIG. 25 shows electrochemistry data in connection with the wild type FAD-GDH protein.

(291) FIG. 26 shows a table of electrochemistry data of the embodiments of the composition of the present invention. Mutations in position 406 provide improved linearity over the entire range of physiological range: F406-S/C/T/V/Y/N/P/L/G/A/I/D/E.

(292) FIG. 27 shows embodiments of the composition of the present invention. Mutations in position 406 that provide improved selectivity of glucose: F406-S/C/T/M/V/Y/N/P/L/G/Q/A/I/D/H/E. F406W provides an example of a substitution that reduces the enzyme selectivity towards glucose. Mutations in position 215 provide improved linearity over the entire range of physiological range: N215-G/H/T/D/Y/S.

(293) An exemplary embodiment of the FAD-GDH composition of the present invention is shown in FIGS. 25A and 25B, showing a graphical representation of the electrochemical response of a mutated FAD-GDH (N177S, N215S, F353L, F406L) to various substrates. FIG. 25A shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus), maltose concentrations (shown as a triangle) and xylose (shown as a rectangle) concentrations (substrate solution were supplemented with 1 mM benzoquinone). R.sup.2 represents a linear fit of the glucose data. FIG. 25B shows a biosensor response to glucose and the non-linear fit through which apparent Km (k) and Imax have been calculated based on Michaelis-Menten equation:

(294) $\begin{array}{cc}I=\frac{I\max \left[S\right]}{\mathrm{Km}+\left[S\right]}& \mathrm{Equation}1\end{array}$
Whereas I is the current, S is the substrate concentration, Imax is the maximum current and the Km is the apparent Michaelis constant.

(295) FIG. 26A shows an exemplary embodiment of a bioelectrochemical response of an FAD-GDH mutant (N177S, N215S, F353L, F406L) of the present invention, varying concentrations of glucose (shown as a rhombus). FIG. 26B shows a bioelectrochemical response of an FAD-GDH mutant (N177S, N215S, F353L, F406L) of the present invention to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. The enzyme activity of FAD-GDH (N177S, N215S, F353L, F406L) was determined by immobilizing FAD-GDH (N177S, N215S, F353L, F406L) to a carbon electrode by an electropolymerization method without the addition of an electron mediator.

(296) FIG. 27A shows an exemplary embodiment of the present invention, showing a biochemical response of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), maltose (shown as a triangle), an xylose (shown as a rectangle). FIG. 27B shows the biochemical response of FAD-GDH (N177S, N215S, F353L, F406L) to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. FAD-GDH (N177S, N215S, F353L, F406L) activity was determined by monitoring a decrease of dichlorophenolindophenol (DCIP) signal at OD.sub.600 (Epoch Microplate Spectrophotometer, Biotek).

(297) FIGS. 28A and 28B, show an electrochemical response to various substrates. In an exemplary embodiment, FIG. 28A shows electrochemical data representing the current response of the biosensor to various glucose concentrations (shown as a rhombus), maltose concentrations (shown as a triangle) and xylose (shown as a rectangle) concentrations. FIG. 28B shows a biosensor response to glucose and the non-linear fit through which apparent K.sub.m (k) and V.sub.max have been calculated. In an exemplary embodiment, wild type FAD-GDH was immobilized to a carbon electrode via an electropolymerization method as described previously. R.sup.2 represents a linear fit of the glucose data. FIG. 28B shows K.sub.mapp=0.85 mM and Imax=1218.5 nA.

(298) FIG. 29A shows biochemical responses of wild type FAD-GDH (w.t.), varying concentrations of glucose (rhombus), maltose (triangle) and xylose (rectangle). FIG. 29B shows a biochemical response of wild type FAD-GDH to glucose and the non-linear fit through which K.sub.m (k) and V.sub.max have been calculated. The enzyme activity of wild type FAD-GDH was determined by monitoring the decrease of Dichlorophenolindophenol (DCIP) signal at OD.sub.600.

(299) Biochemical and Electrochemical comparison between a mutant according to some embodiments of the present invention and FAD-GDH (w.t.), shown in FIGS. 27A and 27B as compared with FIGS. 29A and 29B, demonstrates that the mutant increased in specificity and activity while FIGS. 28A and 28B demonstrate the improvement in bioelectrochemical linearity behavior. Thus, these results distinguish the mutant for glucose-measurements applications over the wild type enzyme.

(300) FIG. 30 is a table showing the results obtained from the biochemical and electrochemical experiments characterizing FAD-GDH (N177S, N215S, F353L, F406L) where: K.sub.m is the Michaelis constant, K.sub.cat is the enzyme's catalytic constant, K.sub.cat/K.sub.m is the catalytic efficiency, glucose/xylose is the ratio of K.sub.catglu/K.sub.catxyl, BEC linearity is the R.sup.2 of the linear fit of the bioelectrochemical experiment, 120 is the current flux (nA/cm.sup.2) measured when 20 mM of glucose was tested, Xylose selectivity is the ratio of I20.sub.glu/I20.sub.xyl, Maltose selectivity is the ratio of I20.sub.glu/I20.sub.mal, pos1-4, the mutated amino acid number.

(301) FIG. 34A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with polypyrrole, and benzoquinone is used as a mediator to obtain a single measurement. FIG. 34B shows the non-linear fit (red line) of the data represented in FIG. 34A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(302) The screen printed electrode (SPE) was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1.2 mg/ml of enzyme and 0.25 M Pyrrole-KCl solution. Electropolymerization was applied via CA 20 pulses of 0.65V, 1 second each with 5 second intervals between each pulse. Glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution, followed by step-wise pipetting of concentrated glucose solution to reach the depicted final glucose solution and by applying 0.2V for 5 minutes at each addition point

(303) FIG. 35A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with polypyrrole, to obtain a single measurement via direct electron transfer. FIG. 35B shows the non-linear fit (red line) of the data represented in FIG. 35A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(304) The screen printed electrode (SPE) was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1.2 mg/ml of enzyme and 0.25 M Pyrrole-KCl solution. Electropolymerization was applied via CA 20 pulses of 0.65V, 1 second each with 5 second intervals between each pulse. Glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution, followed by step-wise pipetting of concentrated glucose solution to reach the depicted final glucose solution and by applying 0.2V for 5 minutes at each addition point.

(305) FIG. 36A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with PEDOT, to obtain a single measurement via direct electron transfer. FIG. 36B shows the non-linear fit (red line) of the data represented in FIG. 36A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(306) The printed electrode (SPE) was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1 mg/ml of enzyme and 0.01 M EDOT/PSS solution (final concentration). Electropolymerization was applied via cycles of CV ranging 0.2-0.85V with a scanning speed of 0.05V/Sec. The immobilized electrode was then washed with DDW and glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution, followed by step-wise pipetting of concentrated glucose solution to reach the depicted final glucose solution and by applying 0.3V for 5 minutes at each addition point.

(307) FIG. 37A shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L) to varying concentrations of glucose (shown as a rhombus), using screen-printed electrodes according to some embodiments of the present invention, where the mutant FAD-GDH protein is immobilized with graphene oxide, to obtain a single measurement via direct electron transfer. FIG. 37B shows the non-linear fit (red line) of the data represented in FIG. 37A. V.sub.max refers to the maximum current flux, K refers to the apparent K.sub.m value extracted from the Michaelis menten equation.

(308) The printed electrode (SPE) was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1 mg/ml of enzyme and 0.01 M EDOT/PSS solution (final concentration). Electropolymerization was applied via cycles of CV ranging 0.2-0.85V with a scanning speed of 0.05V/Sec. The immobilized electrode was then washed with DDW and glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution, followed by step-wise pipetting of concentrated glucose solution to reach the depicted final glucose solution and by applying 0.3V for 5 minutes at each addition point.

(309) FIGS. 38A and 38B shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs (FIG. 38A), or 64 hr (FIG. 38B). In this the mutant FAD-GDH protein is immobilized with polypyrrole, to obtain a single measurement via direct electron transfer.

(310) SPE was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1.2 mg/ml of enzyme and 0.25 M Pyrrole-KCl solution. Electropolymerization was applied via Chronoamperometry (CA) by applying 20 pulses of 0.65V, 1 second each with 5 second intervals between each pulse. Glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution containing 5.5 mM glucose and by applying 0.2V for 20 or 64 hours (FIGS. 38A and 38B, respectively).

(311) FIG. 39 shows an exemplary embodiment of the present invention, showing electrochemistry data of FAD-GDH (N177S, N215S, F353L, F406L), using an electrode according to some embodiments of the present invention configured to measure glucose levels continuously, for 20 hrs. In this embodiment, the mutant FAD-GDH protein is immobilized with PEDOT, to obtain a single measurement via direct electron transfer.

(312) SPE was activated by overlaying it with saturated potassium bicarbonate solution and applying 1.2V for 3 minutes. The electrode was washed 3 times with DDW and overlaid with immobilization solution containing 1 mg/ml of enzyme and 0.01 M EDOT/PSS solution (final concentration). Electropolymerization was applied via cycles of CV ranging 0.2-0.85V with a scanning speed of 0.05V/Sec. Electropolymerization was applied via Chronoamperometry (CA) by applying 20 pulses of 0.65V, 1 second each with 5 second intervals between each pulse. Glucose sensing was conducted by dipping the immobilized enzyme SPE into a PBS solution containing 5.5 mM glucose and by applying 0.3V for 18 hours.

(313) FIG. 40 shows a table of electrochemistry data of the embodiments of the electrodes of the present invention.

(314) While a number of embodiments of the present invention have been described, it is understood that these embodiments are illustrative only, and not restrictive, and that many modifications may become apparent to those of ordinary skill in the art. Further still, the various steps may be carried out in any desired order (and any desired steps may be added and/or any desired steps may be eliminated). All publications and other references mentioned herein are incorporated by reference in their entirety, as if each individual publication or reference were specifically and individually indicated to be incorporated by reference.

(315) Publications and references cited herein are not admitted to be prior art.

REFERENCES

(316) Wang, Joseph (2008). Electrochemical Glucose Biosensors. Chem. Rev. 2008, 108, 814-825. Ferri, Stefano et al. (2011) Review of Glucose Oxidases and Glucose Dehydrogenases: A Bird's Eye View of Glucose Sensing Enzymes. Journal of Diabetes Science and Technology. Volume 5, Issue 5, September 2011.