PQQ-sGDH MUTANT, POLYNUCLEOTIDE AND GLUCOSE DETECTION BIOSENSOR
20190390247 ยท 2019-12-26
Assignee
Inventors
Cpc classification
C12Y101/05002
CHEMISTRY; METALLURGY
International classification
C12Q1/00
CHEMISTRY; METALLURGY
Abstract
The present invention provides a soluble pyrroloquinoline-quinone-dependent glucose dehydrogenase (PQQ-sGDH) mutant, wherein the amino acid positions thereof correspond to a wild-type PQQ-sGDH sequence of Acinetobacter calcoaceticus as shown in SEQ ID NO 1 comprising one of the following group of mutations: A194F, a combined mutation based on A194F, a combined mutation based on Q192A or a combined mutation based on Q192S. Said PQQ-sGDH mutant has good glucose substrate specificity and significantly reduced cross-reactivity to maltose and the like, and is suitable for detecting glucose in a sample such as blood.
Claims
1. A PQQ-sGDH mutant, said PQQ-sGDH derived from a wild-type PQQ-sGDH amino acid sequence SEQ ID NO 1 of Acinetobacter calcoaceticus, or derived from a wild-type amino acid sequence of other microorganisms having at least 90% homology to SEQ ID NO 1, the numbering of amino acid positions of said PQQ-sGDH mutant corresponding to that of the wild-type PQQ-sGDH amino acid sequence SEQ ID NO 1 from A. calcoaceticus, wherein a mutation carried by said PQQ-sGDH mutant comprises: (1) A194F, or (2) one mutation selected from Q192A or Q192S, and said PQQ-sGDH mutant also comprises a substitution mutation occurring at at least one of positions 99, 170, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455.
2. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises: a substitution mutation occurring at at least one of positions 99, 167, 170, 192, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455 of said PQQ-sGDH mutant on the basis of (1); or substitution mutations occurring at at least two of positions 99, 167, 170, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455 of said PQQ-sGDH mutant on the basis of (2).
3. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises a mutation selected from a group consisting of: substitution mutations occurring at at least two of positions 99, 167, 170, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455 of said PQQ-sGDH mutant on the basis of (1); Q192A+G99X+D167X+N452X; Q192S+G99X+D167X+N452X; Q192A+G99X+S170X+Q270X+M365X+N452X+T372X; Q192A+G99X+S170X+Q270X+T366X+N452X+T372X; Q192A+G99X+S170X+Q270X+T366X+N452X+R402X; Q192A+G99X+S170X+Q270X+T366X+N452X+A318X; Q192A+G99X+S170X+Q270X+T366N+K455X+T372X; Q192S+G99X+S170X+Q270X+M365X+N452X+T372X; Q192S+G99X+S170X+Q270X+T366X+N452X+T372X; Q192S+G99X+S170X+Q270X+T366X+N452X+R402X; Q192S+G99X+S170X+Q270X+T366X+N452X+A318X; Q192S+G99X+S170X+Q270X+T366N+K455X+T372X.
4. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises a mutation selected from a group consisting of: A194F+G99X+D167X+N452X; A194F+G99X+S170X+Q270X+T366N+N452X+T372X; A194F+G99X+S170X+Q270X+T366N+N452X+R402X; A194F+G99X+S170X+Q270X+T366N+N452X+A318X; A194F+G99X+S170X+Q270X+T366N+K455X+T372X; Q192A+G99X+D167X+N452X+M365X; Q192A+G99X+D167X+N452X+T366X; Q192A+G99X+S170X+Q270X+M365X+T366X+N452X+T372X; Q192A+G99X+S170X+Q270X+T366N+N452X+K455X+T372X; Q192S+G99X+D167X+N452X+M365X; Q192S+G99X+D167X+N452X+T366X; Q192S+G99X+S170X+Q270X+M365X+T366X+N452X+T372X; and Q192S+G99X+S170X+Q270X+T366N+N452X+K455X+T372X.
5. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises a mutation selected from a group consisting of: A194F+G99X+D167X+N452X+M365X; A194F+G99X+D167X+N452X+T366X; A194F+G99X+S170X+Q270X+T366N+N452X+K455X+T372X; Q192A+G99X+D167X+N452X+T366X+S170X; Q192A+G99X+S170X+Q270X+M365X+T366N+N452X+K455X+T372X; Q192S+G99X+D167X+N452X+T366X+S170X; and Q192S+G99X+S170X+Q270X+M365X+T366N+N452X+K455X+T372X.
6. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises a mutation selected from a group consisting of: A194F+G99X+D167X+N452X+T366X+S170X; Q192A+G99X+D167X+N452X+T366X+S170X+Q270X; and Q192S+G99X+D167X+N452X+T366X+S170X+Q270X.
7. The PQQ-sGDH mutant of claim 1, wherein the mutation carried by said PQQ-sGDH mutant comprises A194F+G99X+D167X+N452X+T366X+S170X+Q270X.
8. The PQQ-sGDH mutant of claim 3, wherein for the mutation carried by said PQQ-sGDH mutant, if G99X is comprised, the X of the G99X is selected from W; if D167X is comprised, the X of the D167X is selected from E; if S170X is comprised, the X of the S170X is selected from G; if Q192X is comprised, the X of the Q192X is selected from A or S; if L193X is comprised, the X of the L193X is selected from P or F; if Q270X is comprised, the X of the Q270X is selected from H; if A318X is comprised, the X of the A318X is selected from D; if M365X is comprised, the X of the M365X is selected from V; if T366X is comprised, the X of the T366X is selected from N or V; if Y367X is comprised, the X of the Y367X is selected from C or S; if T372X is comprised, the X of the T372X is selected from S, G, C or D; if R402X is comprised, the X of the R402X is selected from I; if N452X is comprised, the X of the N452X is selected from T, P, V, C, L, D, I or A; if K455X is comprised, the X of the K455X is selected from R or I.
9. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 1.
10. A biosensor for detecting glucose in a sample, wherein said biosensor comprises said PQQ-sGDH mutant of claim 1.
11. The PQQ-sGDH mutant of claim 4, wherein for the mutation carried by said PQQ-sGDH mutant, if G99X is comprised, the X of the G99X is selected from W; if D167X is comprised, the X of the D167X is selected from E; if S170X is comprised, the X of the S170X is selected from G; if Q192X is comprised, the X of the Q192X is selected from A or S; if L193X is comprised, the X of the L193X is selected from P or F; if Q270X is comprised, the X of the Q270X is selected from H; if A318X is comprised, the X of the A318X is selected from D; if M365X is comprised, the X of the M365X is selected from V; if T366X is comprised, the X of the T366X is selected from N or V; if Y367X is comprised, the X of the Y367X is selected from C or S; if T372X is comprised, the X of the T372X is selected from S, G, C or D; if R402X is comprised, the X of the R402X is selected from I; if N452X is comprised, the X of the N452X is selected from T, P, V, C, L, D, I or A; if K455X is comprised, the X of the K455X is selected from R or I.
12. The PQQ-sGDH mutant of claim 5, wherein for the mutation carried by said PQQ-sGDH mutant, if G99X is comprised, the X of the G99X is selected from W; if D167X is comprised, the X of the D167X is selected from E; if S170X is comprised, the X of the S170X is selected from G; if Q192X is comprised, the X of the Q192X is selected from A or S; if L193X is comprised, the X of the L193X is selected from P or F; if Q270X is comprised, the X of the Q270X is selected from H; if A318X is comprised, the X of the A318X is selected from D; if M365X is comprised, the X of the M365X is selected from V; if T366X is comprised, the X of the T366X is selected from N or V; if Y367X is comprised, the X of the Y367X is selected from C or S; if T372X is comprised, the X of the T372X is selected from S, G, C or D; if R402X is comprised, the X of the R402X is selected from I; if N452X is comprised, the X of the N452X is selected from T, P, V, C, L, D, I or A; if K455X is comprised, the X of the K455X is selected from R or I.
13. The PQQ-sGDH mutant of claim 6, wherein for the mutation carried by said PQQ-sGDH mutant, if G99X is comprised, the X of the G99X is selected from W; if D167X is comprised, the X of the D167X is selected from E; if S170X is comprised, the X of the S170X is selected from G; if Q192X is comprised, the X of the Q192X is selected from A or S; if L193X is comprised, the X of the L193X is selected from P or F; if Q270X is comprised, the X of the Q270X is selected from H; if A318X is comprised, the X of the A318X is selected from D; if M365X is comprised, the X of the M365X is selected from V; if T366X is comprised, the X of the T366X is selected from N or V; if Y367X is comprised, the X of the Y367X is selected from C or S; if T372X is comprised, the X of the T372X is selected from S, G, C or D; if R402X is comprised, the X of the R402X is selected from I; if N452X is comprised, the X of the N452X is selected from T, P, V, C, L, D, I or A; if K455X is comprised, the X of the K455X is selected from R or I.
14. The PQQ-sGDH mutant of claim 7, wherein for the mutation carried by said PQQ-sGDH mutant, if G99X is comprised, the X of the G99X is selected from W; if D167X is comprised, the X of the D167X is selected from E; if S170X is comprised, the X of the S170X is selected from G; if Q192X is comprised, the X of the Q192X is selected from A or S; if L193X is comprised, the X of the L193X is selected from P or F; if Q270X is comprised, the X of the Q270X is selected from H; if A318X is comprised, the X of the A318X is selected from D; if M365X is comprised, the X of the M365X is selected from V; if T366X is comprised, the X of the T366X is selected from N or V; if Y367X is comprised, the X of the Y367X is selected from C or S; if T372X is comprised, the X of the T372X is selected from S, G, C or D; if R402X is comprised, the X of the R402X is selected from I; if N452X is comprised, the X of the N452X is selected from T, P, V, C, L, D, I or A; if K455X is comprised, the X of the K455X is selected from R or I.
15. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 8.
16. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 11.
17. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 12.
18. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 13.
19. An isolated polynucleotide, wherein said polynucleotide encodes said PQQ-sGDH mutant of claim 14.
Description
DETAILED DESCRIPTION
[0040] The present invention obtains a series of PQQ-sGDH mutants by performing a site-directed mutation through PCR amplification, with a gene encoding a wild-type PQQ-sGDH enzyme from A. calcoaceticus LMD79.41 or a DNA from other microorganisms as a template, wherein a protein encoded by the DNA has at least 90% homology to the amino acid sequence of the enzyme. By detecting and comparing the enzymatic activities of the wild-type PQQ-sGDH and various PQQ-sGDH mutants to sugar molecules such as glucose and maltose under the same conditions, the inventors unexpectedly discovered some PQQ-sGDH mutants with superior properties, and these mutants have significantly improved substrate specificity to glucose, and also have significantly reduced cross-reactivity to sugar molecules such as maltose.
[0041] To facilitate calculation and comparison of substrate specificity or cross-reactivity of various PQQ-sGDH mutants and the wild-type PQQ-sGDH, the measured enzymatic activity to glucose as a substrate is defined as 100%, and the measured enzymatic activity to a sugar molecule other than glucose (such as maltose, galactose, etc.) is measured and compared with the former. Based on these measurement results, the substrate specificity or cross-reactivity of various PQQ-sGDH mutants and the wild-type PQQ-sGDH can be evaluated.
[0042] It is known that there are many methods for detecting enzymatic activity of the PQQ-sGDH mutants and the wild-type PQQ-sGDH, for example by detecting their enzymatic activity using reagents PMS and DCIP.
[0043] The percentage of cross-reactivity of the wild-type PQQ-sGDH to a sugar molecule other than glucose is calculated as shown in Equation 1:
cross-reactivity of wild-type PQQ-sGDH [%]=(enzymatic activity of wild-type PQQ-sGDH to sugar molecule other than glucose/enzymatic activity of wild-type PQQ-sGDH to glucose)100%(Equation 1)
[0044] The percentage of cross-reactivity of each of the PQQ-sGDH mutants to a sugar molecule other than glucose is calculated as shown in Equation 2:
cross-reactivity of PQQ-sGDH mutant [%]=(enzymatic activity of PQQ-sGDH mutant to sugar molecule other than glucose/enzymatic activity of PQQ-sGDH mutant to glucose)100%(Equation 2)
[0045] Meanwhile, in order to distinctly show that the cross-reactivity of each PQQ-sGDH mutant with sugar molecules such as maltose and galactose is significantly improved as compared with that of the wild-type PQQ-sGDH, the improvement extent of substrate specificity of each PQQ-sGDH mutant can be calculated according to Equation 3:
[0046] After performing an appropriate mathematical transformation of Equation 3, it can be known that the improved substrate specificity of each PQQ-sGDH mutant is essentially obtained by dividing the calculated value of Equation 1 by the calculated value of Equation 2.
[0047] Other sugar molecule as mentioned in Equations 1, 2, and 3 refers to a sugar molecule other than glucose, which interferes with clinical testing of glucose, and is preferably maltose, galactose, xylose, mannose or allose, particularly maltose or galactose.
[0048] In addition to considering the cross-reactivity and improved substrate specificity of each PQQ-sGDH mutant, another indicator for evaluating the performance of each PQQ-sGDH mutant is the thermal stability of the PQQ-sGDH mutant. That is, the obtained wild-type PQQ-sGDH and each of the PQQ-sGDH mutants are placed at 50 C. for 30 min, and then the initial enzymatic activity before the placement at 50 C. and the residual enzymatic activity after the placement at 50 C. are calculated as percentages.
[0049] The present invention discloses a method for preparing the PQQ-sGDH mutants. During preparation, a series of PQQ-sGDH mutants can be obtained through a PCR site-directed mutation method.
[0050] In order to produce the wild-type PQQ-sGDH and the PQQ-sGDH mutants mentioned above, an expression vector and a host cell can be used, and it should be ensured that the selected expression vector and the selected host cell are matched.
[0051] The expression vector used in the present invention can be divided into a prokaryotic expression vector and a eukaryotic expression vector. For the prokaryotic expression vector and the eukaryotic expression vector, differences in the prokaryotic or eukaryotic cellular hosts determine differences in corresponding expression vectors.
[0052] The prokaryotic cell most commonly used for expression of a foreign protein is Escherichia coli, Bacillus subtilis or the like. The commonly-used expression vector for E. coli is pET series vector (Novagen), pGEX series vector (Pharmacia), pQE series vector (Qiagen), pACYC series vector (Addgene), pBAD series expression vector (Invitrogen), pDEST/pREST series expression vector (Invitrogen), pTrcHis2 series expression vector (Invitrogen) or the like, and the commonly-used expression vector for B. subtilis is pHT01, pHT08, pHT09, pHT10, pHT43 or the like.
[0053] The eukaryotic cell most commonly used for expression of a foreign protein is yeast cell, insect cell, plant cell and mammalian cell. The yeast cell commonly used for expression of the foreign protein is a cell of Pichia pastoris and a cell of Saccharomyces cerevisiae, wherein the commonly-used expression vector for P. pastoris includes pPIC series vector (Invitrogen), pGAPZ/pGAPa series expression vector (Invitrogen) and pAO815 expression vector (Invitrogen); and the commonly-used expression vector for S. cerevisiae includes pYES series expression vector (Invitrogen), pYC series expression vector (Invitrogen) or the like.
[0054] The insect cell commonly used for expression of a foreign protein includes Spodoptera frugiperda cell lines Sf9 and Sf-21, Trichoplusia ni cell lines Tn-368 and BTI-TN-5B1-4, or the like. The expression vector used by these cell lines is a recombinant baculovirus.
[0055] The expression vector commonly used for the plant cell can be selected from derivative vectors constructed based on the Ti plasmid of Agrobacterium tumefaciens or the Ri plasmid of A. rhizogenes, and a plant virus, such as tobacco mosaic virus, potato virus X and cowpea mosaic virus, may also be used as an expression vector.
[0056] The mammalian cell commonly used for expression of a foreign protein is CHO, HEK, BHK, HeLa, COS, SP2/0, NIH3T3, or the like. A common mammalian cell expression vector includes adenovirus expression vector, pSV and pCMV series plasmid vector, poxvirus expression vector, retrovirus expression vector or the like.
[0057] Regardless of which one is selected from the above expression vectors, various methods known in the art, such as calcium chloride transformation, polyethylene glycol (PEG)-mediated protoplast transformation, electroporation, particle bombardment, micro-injection, laser injection, DEAE-dextran transfection, calcium phosphate co-precipitation transfection or artificial liposome-mediated transfection, can be used to introduce such an expression vector carrying a gene encoding a PQQ-sGDH mutant into a suitable host cell, and the resulting host cell is a transformed cell. The transformed cell is cultured under conditions that allow expression of the foreign gene, so that the desired PQQ-sGDH mutant can be produced.
[0058] Of course, a PQQ-sGDH mutant can also be obtained by in vitro translation of mRNA produced by transcription of a gene encoding the mutant, for example by inserting the gene into an appropriate expression vector and the expression vector can be used for an in vitro transcription/translation system.
[0059] Regardless of whether a transformed-cell-based expression system or an in vitro transcription/translation system is employed, after the desired PQQ-sGDH mutant protein is expressed, various conventional protein purification techniques can be used to isolate and purify the mutant, for example, by using chromatography such as ion exchange chromatography, gel filtration chromatography, and affinity chromatography.
[0060] One of the main uses of the series of PQQ-sGDH mutants obtained in the present invention is to be used in a test strip or a biosensor to detect glucose concentration in the blood of a diabetic patient. Of course, in addition to being used for detecting glucose in the blood, it can also be used for detecting glucose in a body fluid such as urine, saliva and tears.
[0061] The present invention also includes a method of detecting glucose in a sample by using a PQQ-sGDH mutant provided by the present invention, and a biosensor and a reagent used in the detection process. There are many methods, biosensors and reagents for detecting glucose by using a PQQ-sGDH enzyme in the art, but only a few examples are given here for illustration, and this does not mean that the methods, biosensors and reagents are limited to these few examples. For example, Chinese patent application CN200580005551.6 disclosed a biosensor for detecting glucose concentration in a sample. A reagent contained in the biosensor can contain a PQQ-sGDH mutant, a stabilizer for stabilizing the enzymatic activity of the PQQ-sGDH mutant, and an electron acceptor, wherein the stabilizer is preferably selected from the group consisting of trehalose, sucrose, glycerol, mannitol and ribose, and the suitable electron acceptor can be selected from potassium ferricyanide, p-benzoquinone and its derivative, phenazine methosulfate (PMS), methyl blue, ferrocene and its derivative. During the detection process, if the sample contains glucose, then the PQQ-sGDH mutant enzymatically reacts with glucose and generates electrons, and the generated electrons are transmitted to an electrode by the electron acceptor, such that the glucose concentration in the sample can be calculated by detecting the generated current value.
[0062] As another example, a method, a device and a reagent disclosed in U.S. Pat. No. 5,484,708 A can also be used for detecting glucose in a sample by steps of: adding 1-naphthol-4-sulfonic acid, a coupling agent, a buffer solution, and a sample solution containing glucose into a cuvette and mixing well; then adding a PQQ-sGDH enzyme to cause an enzymatic reaction which results in a color change; and subsequently measuring the absorbance value at a specific detection wavelength with a spectrophotometer, wherein the specific detection wavelength is related to the selected coupling agent. For example, when the coupling agent is N,N-Bis(2-hydroxyethyl)-4-nitrosoaniline, the detection wavelength is 606 nm, and when the coupling agent is 2,4,6-tribromo-3-hydroxybenzoic acid, the detection wavelength is 705 nm.
[0063] The present invention is further illustrated in the following Examples. These Examples are not intended to limit the scope of the present invention, but to provide a further understanding of the present invention. DNA extraction, cloning, PCR site-directed mutation, construction of a transfer vector, preparation of a transformed cell, protein expression and purification, and the like methods as mentioned in the following Examples are known in the art (Molecular Cloning: A Laboratory Manual (Fourth Edition) edited by Michael R. Green and Joseph Sambrook; Current Protocols in Molecular Biology (2015) edited by Fred M. Ausubel and Roger Bren), and appropriate modifications can be made by those of skills in the art as desired.
Example 1: Cloning and Expression of Wild-Type PQQ-sGDH in E. coli
[0064] A gene encoding the wild-type PQQ-sGDH of A. calcoaceticus strain LMD79.41 was synthesized in vitro by Nanjing GenScript Biotech Co., Ltd. and then the synthesized gene was inserted into a plasmid PET30a (available from Novagen) according to techniques known in the art to obtain a recombinant plasmid. Subsequently, 10 l of the resulting recombinant plasmid was introduced into a host cell of E. coli strain BL21 to obtain E. coli transformed cells, and then after being cultured in 1 ml LB liquid medium at 37 C. for 1 hour, the bacteria were spread on an agar plate and grown overnight at 37 C. Bacterial spots were picked from the agar plate for DNA sequencing. Subsequently, the bacterial spots into which a recombinant plasmid was successfully introduced as confirmed by DNA sequencing were plated to 50 ml LB medium and cultured at 37 C. until the OD.sub.600 was 1.0. 50 l IPTG was added to induce expression of the gene encoding the wild-type PQQ-sGDH, and then after be culturing continually for 3 hours, the bacteria cells were collected.
[0065] These bacterial cells can be collected by centrifugation, and then the recombinant plasmids carrying the gene encoding the wild-type PQQ-sGDH can be isolated from these bacterial cells using QIAGEN Plasmid Midi Kit (Qiagen).
Example 2: Preparation of PQQ-sGDH Mutants Through PCR Site-Directed Mutation
[0066] With the recombinant plasmids isolated in Example 1 as a starting template, A at position 194 was substituted by F through a site-directed mutation carried out by PCR amplification.
[0067] The primers for obtaining the A194F mutation are as shown in SEQ ID NO 3 and SEQ NO 4:
TABLE-US-00001 SEQIDNO3: 5-TGACCAAGGGCGTAACCAGCTTTTCTATTTGTTCTTGCCAAATC AAGCAC-3 SEQIDNO4: 5-GTGCTTGATTTGGCAAGAACAAATAGAAAAGCTGGTTACGCCCT TGGTCA-3.
[0068] With the recombinant plasmids constructed in Example 1 as a starting template, Q at position 192 was substituted by A or S through a site-directed mutation carried out by PCR amplification.
[0069] The primers for obtaining the Q192A mutation are as shown in SEQ ID NO 5 and SEQ NO 6:
TABLE-US-00002 SEQIDNO5: 5-ATTGGTGACCAAGGGCGTAACGCGCTTGCTTATTTGTTCTTGCC AA-3 SEQIDNO6: 5-TTGGCAAGAACAAATAAGCAAGCGCGTTACGCCCTTGGTCACCA AT-3.
[0070] The primers for obtaining the Q192S mutation are as shown in SEQ ID NO 7 and SEQ NO 8:
TABLE-US-00003 SEQIDNO7: 5-TGGTGACCAAGGGCGTAACTCGCTTGCTTATTTGTTCTTG CCA-3 SEQIDNO8: 5-TGGCAAGAACAAATAAGCAAGCGAGTTACGCCCTTGGTCA CCA-3.
[0071] A PCR reaction was carried out by using the aforementioned primers according to the instructions of Primerstar polymerase purchased from Takara Co., Ltd. to respectively obtain amplified products of the PQQ-sGDH mutant-encoding genes in which a single-point mutation (A194F, Q192A or Q192S) occurred. Then cloning and expression, culturing and collecting of bacterial cells were carried out according to the method of Example 1, and meanwhile the recombinant plasmids carrying the PQQ-sGDH mutant-encoding gene in which the single-point mutation occurred were obtained from the collected bacterial cells.
[0072] Next, a PCR site-directed mutation was conducted again by using the obtained recombinant plasmids carrying the PQQ-sGDH mutant encoding gene in which the single-point mutation occurred, such that one mutation selectively occurred at one of positions 99, 167, 170, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455 on the basis of A194F, Q192A or Q192S, thereby obtaining a series of combined mutations at two positions based on A194F, Q192A or Q192S. In particular, a saturation mutation was also performed on position 452. That is, N at the position 452 was substituted by A, R, D, C, Q, E, H, I, G, L, K, M, F, P, S, T, W, Y or V respectively. If it is wanted to obtain combined mutations at three positions, a mutation should occur selectively at another one of positions 99, 167, 170, 193, 270, 318, 365, 366, 367, 372, 402, 452 and 455 based on the combined mutations at two positions. Similarly, by repeating the above operation, combined mutations at four or more positions based on A194F, Q192A or Q192S can also be obtained. For each of the combined mutations described above, cloning and expression, and culturing and collecting of bacterial cells were carried out according to the method of Example 1, and meanwhile the corresponding recombinant plasmids were also obtained from the collected bacterial cells.
[0073] All of the above PQQ-sGDH mutant-encoding genes were verified by DNA sequencing.
Example 3: Purification of Wild-Type PQQ-sGDH or PQQ-sGDH Mutant
[0074] The bacterial cells collected in Example 1 (carrying the wild-type PQQ-sGDH encoding gene) or the bacterial cells collected in Example 2 (carrying the PQQ-sGDH mutant encoding gene) were first cultured at 37 C. until the OD.sub.600 was 1.0, and then 50 l IPTG was added to induce expression of the gene encoding the wild-type PQQ-sGDH or the PQQ-sGDH mutant. Subsequently, E. coli cells were collected from the medium by centrifugation and resuspended with phosphate buffer A (0.2M sodium chloride, 20 mM sodium dihydrogen phosphate, pH 7.0). Then the wild-type PQQ-sGDH or the PQQ-sGDH mutant contained in the cells was released by high pressure, ultrasonic or the like disruption methods, and the supernatant is collected by centrifugation, such that the obtained supernatant contains a solution of crude enzyme of the wild-type PQQ-sGDH or the PQQ-sGDH mutant.
[0075] Next, the obtained crude enzyme solution sample was loaded onto a His-tag adsorption column previously equilibrated with phosphate buffer A, and then the column was first washed with phosphate buffer B (0.2M sodium chloride, 50 mM imidazole, and 20 mM sodium dihydrogen phosphate, pH 7.0) and finally eluted with phosphate buffer C (0.5M sodium chloride, 150 mM imidazole, 20 mM sodium dihydrogen phosphate, pH 7.0), so that a high-purity enzyme solution of the wild-type PQQ-sGDH or the PQQ-sGDH mutant was obtained. The resulting enzyme solution can also be made into an enzyme powder after being dried.
Example 4: Enzymatic Activity Detection of Wild-Type PQQ-sGDH and PQQ-sGDH Mutant
[0076] Reagent preparation was required before detection: reagent A: 10 mM MOPS buffer (MOPS-NaOH buffer solution containing 1 mM CaCl.sub.2, at pH 7.0); reagent B: glucose solution (1.2M); reagent C: PMS solution (20 mM); reagent D: DCIP solution (4.0 mM); and reagent E: enzyme dilution (20 mM MOPS-NaOH buffer containing 1 mM CaCl.sub.2 and 0.1% Triton X-100, pH 7.0).
[0077] A suitable amount of PQQ was added to the reagent A until the concentration of PQQ reached 1 mM, so as to obtain a reagent A1. The powder of the wild-type PQQ-sGDH or the PQQ-sGDH mutant in Example 3 was dissolved in deionized water until its concentration was 1 mg/ml, such that a detection enzyme solution was obtained.
[0078] In order to measure the enzymatic activity, the wild-type PQQ-sGDH or the PQQ-sGDH mutant was first activated, namely 900 l reagent A1 and 100 l detection enzyme solution reacted for 1 hour at room temperature for enzyme activation.
[0079] After the enzymatic activation, the enzymatic activity of the wild-type PQQ-sGDH or the PQQ-sGDH mutant was detected and the method for detecting the enzymatic activity was as follows, wherein the detection principle of the method was:
PQQ-sGDH+glucose+PMS.fwdarw.glucono-1,5-lactone+PMS(reduced form)
PMS(reduced form)+DCIP.fwdarw.PMS+DCIP(reduced form)
[0080] After the reaction was completed, the absorbance value (OD) at a wavelength of 600 nm was measured using a spectrophotometer. Definition: the enzymatic activity of reducing 1 mol of DCIP by the wild-type PQQ-sGDH or the PQQ-sGDH mutant in 1 minute was 1 U.
[0081] The detailed detecting steps were as follows: [0082] (1) The following reaction mixture was prepared in a lightproof reaction flask and stored on ice (for temporary preparation) with the following components: [0083] 2.5 ml MOPS-NaOH buffer solution (pH 7.0) concentration after mixing (17 mM) [0084] 0.3 ml glucose solution concentration after mixing (124 mM) [0085] 0.5 ml PMS solution concentration after mixing (3.45 mM) [0086] 0.5 ml DCIP solution concentration after mixing (0.69 mM) [0087] (2) 2.9 ml of the reaction mixture was added into a test tube, placed in a water bath kettle at 25 C. and preheated for 5 min; [0088] (3) 0.1 ml of the enzyme solution of the wild-type PQQ-sGDH or the PQQ-sGDH mutant was added, and a reverse mixing was conducted smoothly; [0089] (4) The decline curve of OD at the wavelength of 600 nm within 5 min was plotted using a spectrophotometer under conditions of a constant temperature at 25 C. The OD change per 1 minute (OD.sub.test) was calculated according to the straight-line portion at the beginning of the curve, and meanwhile the enzyme solution in (4) was replaced with the reagent E and a blank value (OD.sub.blank) was determined according to the same method; [0090] (5) The enzymatic activity was calculated:
U/ml=[OD/min(OD.sub.testOD.sub.blank)Vtdf]/(221.0Vs),
U/mg=(U/ml)1/C, wherein,
[0091] Vt is the total reaction volume of 3.0 ml, and Vs is the sample volume of 0.1 ml; 22: the absorption coefficient in millimole (cm.sup.2/micromole) of DCIP under the above detection conditions; 1.0: optical path; df: dilution factor, and C: enzyme concentration in a solution (mg/ml).
[0092] It should be noted that, the enzyme powder should be dissolved in the pre-cooled reagent E and diluted with the same buffer to 0.1-0.8 U/ml before testing. Moreover, a plastic tube is preferred for use in the detection due to the adhesivity of the enzyme.
Example 5: Determination of Thermal Stability of Wild-Type PQQ-sGDH or PQQ-sGDH Mutant
[0093] A 20 mM potassium phosphate solution (pH 7.0) containing 1 mg of the wild-type PQQ-sGDH or the PQQ-sGDH mutant and 0.016 mg PQQ was prepared to activate the wild-type PQQ-sGDH or the PQQ-sGDH mutant. After incubation at room temperature for 30 min, the initial enzyme activity to glucose was measured according to the method of Example 4, and then after the enzyme solution was incubated in a water bath at 50 C. for 30 minutes, the residual enzyme activity of the enzyme solution was detected and calculated as a percentage.
Example 6: Cross-Reactivity and Thermal Stability Tests of Wild-Type PQQ-sGDH and PQQ-sGDH Mutant
[0094] The bacterial cells collected in Example 1 or Example 2 were subjected to cell disruption by sonication. The disrupted bacterial solution was centrifuged, 100 l of the supernatant after centrifugation was taken, and 900 l of 10 mM MOPS buffer (pH 7.0) containing 1 mM PQQ and 1 mM CaCl.sub.2 was added into the supernatant to react for 1 hour at room temperature. The holoenzyme formed after the reaction was used for the following cross-reactivity and thermal stability tests.
[0095] According to the enzymatic-activity determining method in Example 4, a certain amount of enzyme solution of the wild-type PQQ-sGDH and the PQQ-sGDH mutant was respectively taken and tested with an aqueous glucose solution having a concentration of 30 mM, and meanwhile was respectively tested with an aqueous maltose solution having a concentration of 30 mM.
[0096] During calculation of the enzymatic activity, the value obtained when 30 mM glucose was used as the substrate was set as 100% activity. The measured value for maltose was compared with the value for glucose, and the cross-reactivity [%] of the wild-type PQQ-sGDH to maltose and the cross-reactivity [%] of the PQQ-sGDH mutant to maltose were calculated according to Equation 1 and Equation 2 respectively. In the tables below, the cross-reactivity of the wild-type PQQ-sGDH or the PQQ-sGDH mutant to maltose was expressed in M/G. At the same time, based on this, the improved substrate specificity of each PQQ-sGDH mutant was calculated according to Equation 3.
[0097] The thermal stabilities of the wild-type PQQ-sGDH and the PQQ-sGDH mutants were tested as in Example 5.
(1) A194F and A194F-Based Combined Mutation
[0098] The test results of cross-reactivity and thermal stability of them were shown in Table 1:
TABLE-US-00004 TABLE 1 Improved Relative Thermal Substrate M/G Activity Stability Specificity Wild-type PQQ-sGDH Wild Type 104.00% 100.00% 90.00% N/A PQQ-sGDH mutant (A194F mutation at single position) A194F 86.46% 17.87% 92.20% 1.20 PQQ-sGDH mutant (A194F-based combined mutation at two positionss) A194F + L193P 22.11% N/A N/A 4.70 A194F + N452P 35.52% N/A N/A 2.93 A194F + M365V 35.00% N/A N/A 2.97 A194F + T366N 23.44% N/A N/A 4.44 A194F + G99W 30.75% N/A N/A 3.38 A194F + D167E 31.48% N/A N/A 3.30 A194F + S170G 27.07% N/A N/A 3.84 A194F + Q270H 24.00% N/A N/A 4.33 A194F + T372S 36.00% N/A N/A 2.89 A194F + T372G 26.00% N/A N/A 4.00 A194F + T372C 31.70% 46.29% 91.25% 3.28 A194F + T372D 29.64% 44.30% 89.02% 3.51 A194F + R402I 43.10% N/A N/A 2.41 A194F + Q192A 14.75% N/A N/A 7.05 A194F + Q192S 12.24% N/A N/A 8.50 PQQ-sGDH mutant (A194F-based combined mutation at three positions) A194F + N452P + M365V 27.76% N/A N/A 3.75 A194F + N452T + T366N 15.31% N/A N/A 6.79 A194F + K455I + D167E 26.14% N/A N/A 3.98 A194F + N452T + Y367C 23.19% N/A N/A 4.48 A194F + N452P + D167E 20.88% N/A N/A 4.98 A194F + D167E + S170G 22.31% N/A N/A 4.66 A194F + G99W + Q270H 23.33% N/A N/A 4.46 A194F + N452P + A318D 12.39% N/A N/A 8.39 A194F + N452P + R402I 27.82% N/A N/A 3.74 A194F + Q192S + N452P 10.23% N/A N/A 10.17 A194F + Q192A + N452P 8.98% N/A N/A 11.58 A194F + D167E + N452P 15.75% N/A N/A 6.60 PQQ-sGDH mutant (A194F-based combined mutation at four or more positions) A194F + D167E + N452P + G99W 8.09% N/A N/A 12.86 A194F + D167E + N452P + G99W + M365V 6.43% N/A N/A 16.17 A194F + D167E + N452P + G99W + T366N 3.06% N/A N/A 33.99 A194F + D167E + N452P + G99W + T366N + S170G 4.52% N/A N/A 23.01 A194F + D167E + N452P + G99W + T366N + S170G + Q270H 3.11% N/A N/A 33.44 A194F + N452P + G99W + T366N + S170G + Q270H + T372S 3.16% N/A N/A 32.91 A194F + N452P + G99W + T366N + S170G + Q270H + T372G 2.08% N/A N/A 50.00 A194F + N452A + G99W + T366N + S170G + Q270H + R402I 4.55% N/A N/A 22.86 A194F + N452A + G99W + T366N + S170G + Q270H + A318D 3.41% N/A N/A 30.50 A194F + N452T + G99W + T366N + S170G + Q270H + T372S 2.32% N/A N/A 44.83 A194F + K455I + G99W + T366N + S170G + Q270H + T372G 2.48% N/A N/A 41.94 A194F + N452T + K455I + G99W + T366N + S170G + Q270H + T372G 1.87% N/A N/A 55.62
[0099] As could be seen from Table 1, the M/G for the single-position mutation A194F of the PQQ-sGDH mutant was 86.46% and the improved substrate specificity was 1.20, which meant that the mutant had improved substrate specificity (maltose/glucose) 1.20 times larger than that of the wild-type PQQ-sGDH. Also, the lowest M/G of the A194F-based combined mutations at two positions was 12.24%, and the lowest improved substrate specificity thereof was 2.41. Also, the lowest M/G of the A194F-based combined mutations at three positions was 8.98%, and the lowest improved substrate specificity thereof was 3.74. Also, the lowest M/G of the A194F-based combined mutations at four or more positions was 1.87%, and the lowest improved substrate specificity thereof was 12.86. It could be seen that each of the PQQ-sGDH mutants listed in Table 1 had significantly reduced cross-reactivity to maltose, and significantly increased substrate specificity to glucose.
(2) Q192A-Based Combined Mutation
[0100] The test results of cross-reactivity and thermal stability of them were shown in Table 2:
TABLE-US-00005 TABLE 2 Improved Relative Thermal Substrate M/G Activity Stability Specificity Wild-type PQQ-sGDH Wild Type 104.00% 100.00% 90.00% N/A PQQ-sGDH mutant (Q192A-based combined mutation at two positions) Q192A + L193P 26.98% 44.77% 87.55% 3.85 Q192A + N452T 32.91% 60.50% 81.23% 3.16 Q192A + N452P 22.34% 71.58% 84.78% 4.66 Q192A + N452V 21.25% 51.37% N/A 4.89 Q192A + N452C 33.04% 63.30% 87.54% 3.15 Q192A + N452L 31.27% N/A N/A 3.33 Q192A + N452D 36.12% N/A N/A 2.88 Q192A + N452I 40.01% N/A N/A 2.60 Q192A + N452A 32.88% N/A N/A 3.16 Q192A + K455R 40.55% N/A N/A 2.56 Q192A + K455I 39.21% N/A N/A 2.65 Q192A + M365V 25.76% 43.19% N/A 4.04 Q192A + T366N 14.36% 56.52% 99.22% 7.24 Q192A + T366V 16.27% 54.14% 89.19% 6.39 Q192A + Y367C 19.49% N/A N/A 5.34 Q192A + Y367S 27.57% N/A N/A 3.77 Q192A + G99W 18.73% N/A N/A 5.55 Q192A + D167E 17.80% 42.53% 74.60% 5.84 Q192A + S170G 25.01% 36.45% N/A 4.16 Q192A + Q270H 38.37% 55.33% N/A 2.71 Q192A + R402I 40.11% N/A N/A 2.59 Q192A + T372S 20.48% 45.10% 39.46% 5.08 Q192A + T372C 23.30% 30.51% 63.90% 4.46 Q192A + T372D 18.73% 30.30% 67.55% 5.56 Q192A + T372G 15.31% 37.84% 30.54% 6.79 PQQ-sGDH mutant (Q192A-based combined mutations at three positions) Q192 A + N452P + M365V 25.11% N/A N/A 4.14 Q192A + D167E + T366N 9.04% N/A N/A 11.50 Q192A + N452P + T366V 12.99% N/A N/A 8.02 Q192A + N452P + Y367C 17.76% N/A N/A 5.86 Q192A + K455I + Y367S 20.43% N/A N/A 5.09 Q192A + N452V + T372C 11.42% N/A N/A 9.11 Q192A + N452T + T372D 12.42% N/A N/A 8.37 Q192A + N452P + T372S 14.05% N/A N/A 7.40 Q192A + N452P + D167E 20.67% N/A N/A 5.03 Q192A + D167E + S170G 9.73% N/A N/A 10.69 Q192A + N452P + Q270H 16.65% N/A N/A 6.25 Q192A + N452T + A318D 17.72% N/A N/A 5.87 Q192A + N452T + G99W 6.74% N/A N/A 15.43 Q192A + N452T + R402I 17.38% N/A N/A 5.984 Q192A + D167E + N452P 6.89% N/A N/A 15.09 PQQ-sGDH mutant (Q192A-based combined mutation at four positions) Q192A + D167E + N452P + G99W 5.98% N/A N/A 17.39 Q192A + D167E + N452P + G99W + M365V 4.37% N/A N/A 23.80 Q192A + D167E + N452P + G99W + T366N 4.51% N/A N/A 23.06 Q192A + D167E + N452P + G99W + T366N + S170G 4.03% N/A N/A 25.81 Q192A + D167E + N452P + G99W + T366N + S170G + Q270H 3.51% N/A N/A 29.63 Q192A + N452P + G99W + T366N + S170G + Q270H + T372S 7.11% N/A N/A 14.63 Q192A + N452P + G99W + M365V + S170G + Q270H + T372S 8.89% N/A N/A 11.70 Q192A + N452P + G99W + M365V + T366N + S170G + Q270H + T372S 6.15% N/A N/A 16.91 Q192A + N452P + G99W + T366N + S170G + Q270H + T372G 4.65% N/A N/A 22.37 Q192A + N452A + G99W + T366N + S170G + Q270H + R402I 5.15% N/A N/A 20.19 Q192A + N452A + G99W + T366N + S170G + Q270H + A318D 5.62% N/A N/A 18.51 Q192A + N452T + G99W + T366N + S170G + Q270H + T372S 2.37% N/A N/A 43.88 Q192A + K455I + G99W + T366N + S170G + Q270H + T372G 2.09% N/A N/A 49.76 Q192A + N452T + K455I + G99W + T366N + S170G + Q270H + T372G 1.99% N/A N/A 52.26 Q192A + N452P + K455R + G99W + M365V + T366N + S170G + Q270H + T372S 2.67% N/A N/A 38.95
[0101] As could be seen from Table 2, the lowest M/G of the Q192A-based combined mutations at two positions was 14.36%, and the lowest improved substrate specificity thereof was 2.56. Also, the lowest M/G of the Q192A-based combined mutations at three positions was 6.74%, and the lowest improved substrate specificity thereof was 4.14. Also, the lowest M/G of the Q192A-based combined mutations at four or more positions was 1.99%, and the lowest improved substrate specificity thereof was 11.70. It could be seen that each of the PQQ-sGDH mutants listed in Table 2 had significantly reduced cross-reactivity to maltose, and significantly increased substrate specificity to glucose.
(3) Q192S-Based Combined Mutation
[0102] The test results of cross-reactivity and thermal stability of them were shown in Table 3:
TABLE-US-00006 TABLE 3 Improved Relative Thermal Substrate M/G Activity Stability Specificity Wild-type PQQ-sGDH Wild Type 104.00% 100.00% 90.00% N/A PQQ-sGDH mutant (Q192S-based combined mutation at two positions) Q192S + L193P 25.66% 48.98% 90.80% 4.05 Q192S + N452T 29.13% 61.77% 77.41% 3.57 Q192S + N452P 20.41% 77.53% 86.12% 5.10 Q192S + N452V 19.98% 57.49% N/A 5.21 Q192S + N452C 30.93% 67.41% 88.91% 3.36 Q192S + N452L 36.43% N/A N/A 2.85 Q192S + N452D 32.97% N/A N/A 3.15 Q192S + N452I 36.20% N/A N/A 2.87 Q192S + N452A 31.16% N/A N/A 3.34 Q192S + K455R 37.23% N/A N/A 2.79 Q192S + K455I 36.83% N/A N/A 2.82 Q192S + M365V 21.65% 49.14% N/A 4.80 Q192S + T366N 9.55% 62.74% 91.24% 10.89 Q192S + T366V 14.12% 53.16% 79.97% 7.37 Q192S + Y367C 17.84% N/A N/A 5.83 Q192S + Y367S 24.10% N/A N/A 4.32 Q192S + G99W 17.71% N/A N/A 5.87 Q192S + D167E 16.65% 48.77% 69.48% 6.25 Q192S + S170G 23.13% 44.37% N/A 4.50 Q192S + Q270H 34.92% 60.48% N/A 2.98 Q192S + R402I 38.44% N/A N/A 2.71 Q192S + T372S 16.22% 49.32% 40.85% 6.41 Q192S + T372G 14.10% 44.29% 33.77% 7.38 Q192S + T372C 14.33% 60.72% 82.65% 7.26 Q192S + T372D 15.63% 61.84% 88.04% 6.65 PQQ-sGDH mutant (Q192S-based combined mutation at three positions) Q192S + D167E + M365V 23.88% N/A N/A 4.36 Q192S + N452P + T366N 8.23% N/A N/A 12.64 Q192S + N452T + T366V 12.87% N/A N/A 8.08 Q192S + K455I + Y367C 16.54% N/A N/A 6.29 Q192S + N452T + Y367S 19.76% N/A N/A 5.26 Q192S + N452V + T372S 10.28% N/A N/A 10.12 Q192S + N452T + T372D 11.87% N/A N/A 8.76 Q192S + N452P + T372C 12.73% N/A N/A 8.17 Q192S + N452P + D167E 6.67% N/A N/A 15.59 Q192S + D167E + S170G 10.37% N/A N/A 10.29 Q192S + N452P + Q270H 15.42% N/A N/A 6.74 Q192S + G99W + A318D 17.23% N/A N/A 6.04 Q192S + N452T + G99W 6.63% N/A N/A 15.69 Q192S + N452T + R402I 15.23% N/A N/A 6.83 Q192S + D167E + N452P 9.06% N/A N/A 11.48 PQQ-sGDH mutant (Q192S-based combined mutation at four or more positions) Q192S + D167E + N452P + G99W 6.95% N/A N/A 14.96 Q192S + D167E + N452P + G99W + M365V 4.50% N/A N/A 23.11 Q192S + D167E + N452P + G99W + T366N 5.88% N/A N/A 17.69 Q192S + D167E + N452P + G99W + T366N + S170G 6.29% N/A N/A 16.53 Q192S + D167E + N452P + G99W + T366N + S170G + Q270H 4.76% N/A N/A 21.85 Q192S + N452P + G99W + T366N + S170G + Q270H + T372S 6.40% N/A N/A 16.25 Q192S + N452P + G99W + M365V + S170G + Q270H + T372S 8.15% N/A N/A 12.76 Q192S + N452P + G99W + M365V + T366N + S170G + Q270H + T372S 5.87% N/A N/A 17.72 Q192S + N452P + G99W + T366N + S170G + Q270H + T372G 4.87% N/A N/A 21.36 Q192S + N452A + G99W + T366N + S170G + Q270H + R402I 6.23% N/A N/A 16.69 Q192S + N452A + G99W + T366N + S170G + Q270H + A318D 6.90% N/A N/A 15.07 Q192S + N452T + G99W + T366N + S170G + Q270H + T372S 3.13% N/A N/A 33.23 Q192S + K455I + G99W + T366N + S170G + Q270H + T372G 2.96% N/A N/A 35.14 Q192S + N452T + K455I + G99W + T366N + S170G + Q270H + T372G 1.75% N/A N/A 59.43 Q192S + N452P + K455R + G99W + M365V + T366N + S170G + Q270H + T372S 2.33% N/A N/A 44.64
[0103] As could be seen from Table 3, the lowest M/G of the Q192S-based mutations at two positions was 9.55%, and the lowest improved substrate specificity thereof was 2.98. Also, the lowest M/G of the Q192S-based combined mutations at three positions was 6.63%, and the lowest improved substrate specificity thereof was 4.36. Also, the lowest M/G of the Q192S-based combined mutations at four or more positions was 1.75%, and the lowest improved substrate specificity thereof was 12.76. It could be seen that each of the PQQ-sGDH mutants listed in Table 3 had significantly reduced cross-reactivity to maltose, and significantly increased substrate specificity to glucose.
Example 7: Maltose Interference Experiment
[0104] A glucose assay was performed with a wild-type PQQ-sGDH or a PQQ-sGDH mutant. The reference sample contained 50 mg/dl glucose. The test sample contained 50 mg/dl glucose and 100 mg/dl maltose. The same amount of enzymatic activity (U/ml; see Example 4) was used for each assay.
[0105] The test solution was prepared in advance:
1000 l of 0.2M citric acid buffer (pH 5.8) containing 0.315 mg of (4-(dimethylphosphinylmethyl)-2-methylpyrazolo-[1.5a]-imidazol-3-yl)-(4-nitrosophenyl)-amine).
[0106] Next, 1 ml of the test solution and 0.015 ml of the reference sample or the test sample were added into a cuvette, and mixed well.
[0107] After adding 0.050 ml of 90 U/ml wild-type PQQ-sGDH or PQQ-sGDH mutant, this assay then started. The absorbance change at 620 nm was monitored. A constant value was observed after 5 min, and then the absorbance change value within 5 min was calculated. The absorbance change value obtained by measuring the reference sample with the wild-type PQQ-sGDH was set as 100%, and then the percentage of the wild-type PQQ-sGDH for measuring the test sample and the percentages of the PQQ-sGDH mutant for measuring the reference sample and the test sample were respectively calculated based on this set value. The results were shown in Table 4:
TABLE-US-00007 TABLE 4 50 mg/dl glucose and 100 50 mg/dl glucose mg/dl maltose Wild-type PQQ-sGDH 100% 160% PQQ-sGDH mutant 100% 121% (A194F)
[0108] It could be seen clearly from Table 4 that, the measured glucose value was significantly decreased when the PQQ-sGDH mutant (A194F) was used in the assay, which meant that as compared with the wild-type PQQ-sGDH, when the PQQ-sGDH mutant (A194F) was used for measuring glucose, the interference of the presence of maltose to the measurement results was significantly reduced.
[0109] Maltose interference experiments were conducted with other PQQ-sGDH mutants of the present invention, it was also found that the interference of the presence of maltose to the measurement results was significantly reduced as compared with the wild-type PQQ-sGDH.
[0110] Furthermore, a glucose detection biosensor disclosed in Chinese patent application CN200580005551.6 could also be used to detect the concentration of glucose in a blood sample, wherein the reaction layer of the biosensor contained the PQQ-sGDH mutant provided by the present invention, and the results meant that if maltose was present in the sample, the accuracy of numerical values detected with any one of the PQQ-sGDH mutants provided by the present invention was significantly increased as compared to the wild-type PQQ-sGDH, and the interference of the maltose in the sample to the measurement results was significantly reduced.