The present invention provides a method for changing an coenzyme activity and a preference of glucose dehydrogenase and the use thereof, and relates to the technical field of genetic engineering. In the method of the present invention, by performing a site-directed mutation of the fourth amino acid in a conserved sequence GXXXGXG of the glucose dehydrogenase, a mutant protein with the changed coenzyme activity and preference can be simply and directly obtained without a large amount of screening, such that the enzyme can be widely applied to catalyze a coenzyme regeneration reaction. In the embodiments of the present invention, four glucose dehydrogenase mutants T17G, T17K, T17RGDH DN46 and K17G are finally obtained by site-directed mutation of the 4th amino acid (which binds with a nicotinamide coenzyme ribose 2-phosphate) in the conserved sequence GXXXGXG widely existed in glucose dehydrogenases from different sources, and upon verification, the enzyme activity, preference and the like of them all have been varied.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

In some embodiments, the present invention provides a protein comprising amino acids in the following sequence L(X).sub.n=14X.sup.3(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=32-34X.sup.2 (SEQ ID NO: 128), wherein each X independently represents any naturally occurring amino acid residue and n indicates the number of amino acid residues represented by the respective paranthetical 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.3 is D; and/or b) X.sup.2 is selected from the group consisting of H, L, S or V. In some embodiments, the present invention also provides a protein comprising amino acids in the sequence set forth by SEQ ID NO: 38 or SEQ ID NO: 39, except that: the amino acid at position 406 is an amino acid other than F; and/or the amino acid at position 474 is an amino acid other than N.

A glucose monitoring method and a glucose sensor, both of which use glucose dehydrogenase having a Michaelis constant (Km) for xylose of 600 mM or more and 3000 mM or less, and a Km for glucose of 0.1 mM or more and 100 mM or less, which provide for evaluating FADGDH in an aqueous system while reducing the practical influence of FADGDH on D-xylose.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.

The present invention provides members that produce on a large scale a coenzyme-linked glucose dehydrogenase which has excellent substrate-recognizing ability toward glucose while providing low action on maltose. The present invention relates to a polynucleotide encoding a soluble coenzyme-linked glucose dehydrogenase that catalyzes the oxidation of glucose in the presence of an electron acceptor and has an activity toward maltose of 5% or lower; a polypeptide encoded by the nucleotide sequence of the polynucleotide; a recombinant vector carrying the polynucleotide; a transformed cell produced using the recombinant vector; a method for producing a polypeptide comprising culturing the transformed cell and collecting from the cultivated products a polypeptide that links to FAD to exert the glucose dehydration activity; a method for determination of glucose using the polypeptide; a reagent composition for determination of glucose; and a biosensor.