FAD-CONJUGATED GLUCOSE DEHYDROGENASE GENE
An object of the present invention is to provide: a novel gene (polynucleotide) encoding an FAD-conjugated glucose dehydrogenase having excellent properties that it has excellent reactivity to glucose, excellent thermal stability, and excellent substrate-recognition performance and also has a low activity for maltose; a process for the production of the enzyme using a transformant cell transfected with the gene; and a method for the determination of glucose, a reagent composition for use in the determination of glucose, a biosensor for use in the determination of glucose and others, each characterized by using the enzyme obtained. The invention relates to a polynucleotide encoding an FAD-conjugated glucose dehydrogenase, comprising a polypeptide containing an amino acid sequence: X1-X2-X3-X4-X5-X6 (wherein X1 and X2 independently represent an aliphatic amino acid residue; X3 and X6 independently represent a branched amino acid residue; and X4 and X5 independently represent a heterocyclic amino acid residue or an aromatic amino acid residue); and others.
1 - 24 . (canceled)
25 . A biosensor comprising a recombinant FAD-conjugated glucose dehydrogenase,
wherein the recombinant FAD-conjugated glucose dehydrogenase is a polypeptide comprising amino acid sequence AGVPWV and is selected from:
(a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1; and
(b) a polypeptide which comprises an amino acid sequence having a homology of 90% or more to the amino acid sequence (a) and has an FAD-conjugated glucose dehydrogenase activity, and wherein the biosensor is capable of detecting glucose.
26 . The biosensor of claim 25 , wherein the biosensor comprises a reaction layer comprising the recombinant FAD-conjugated glucose dehydrogenase.
27 . The biosensor of claim 26 , wherein the reaction layer further comprises a hydrophilic polymer and an electron acceptor.
28 . The biosensor of claim 27 , wherein the reaction layer is in contact with an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate.
29 . The biosensor of claim 25 , which is capable of detecting glucose by detection of an oxidation current.
30 . The biosensor of claim 25 , which is capable of detecting glucose by a change in color intensity.
31 . The biosensor of claim 25 , which is capable of detecting glucose by a pH change.
32 . A method of making the biosensor of claim 25 , comprising: forming an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate, and forming a reaction layer on the electrode system comprising a hydrophilic polymer, the recombinant FAD-conjugated glucose dehydrogenase, and an electron receptor.
33 . A biosensor comprising a recombinant FAD-conjugated glucose dehydrogenase,
wherein the recombinant FAD-conjugated glucose dehydrogenase is a polypeptide comprising amino acid sequence AGVPWV and is selected from:
(a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1; and
(b) a polypeptide which comprises an amino acid sequence having a homology of 90% or more to the amino acid sequence (a) and has an FAD-conjugated glucose dehydrogenase activity;
wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 10% or less with a value of enzymatic activity for D-glucose taken as 100%; and
wherein the biosensor is capable of detecting glucose.
34 . The biosensor of claim 33 , wherein the biosensor comprises a reaction layer comprising the recombinant FAD-conjugated glucose dehydrogenase.
35 . The biosensor of claim 34 , wherein the reaction layer further comprises a hydrophilic polymer and an electron acceptor.
36 . The biosensor of claim 35 , wherein the reaction layer is in contact with an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate.
37 . The biosensor of claim 33 , which is capable of detecting glucose by detection of an oxidation current.
38 . The biosensor of claim 33 , which is capable of detecting glucose by a change in color intensity.
39 . The biosensor of claim 33 , which is capable of detecting glucose by a pH change.
40 . The biosensor of claim 33 , wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 5% or less with a value of enzymatic activity for D-glucose taken as 100%.
41 . The biosensor of claim 40 , wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 3% or less with a value of enzymatic activity for D-glucose taken as 100%.
42 . A biosensor comprising a recombinant FAD-conjugated glucose dehydrogenase,
wherein the recombinant FAD-conjugated glucose dehydrogenase is a polypeptide comprising amino acid sequence AGVPWV and is selected from:
(a) a polypeptide which comprises the amino acid sequence represented by SEQ ID NO: 1; and
(b) a polypeptide which comprises an amino acid sequence having a homology of 90% or more to the amino acid sequence (a) and has an FAD-conjugated glucose dehydrogenase activity;
wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 10% or less with a value of enzymatic activity for D-glucose taken as 100%, and
wherein the biosensor can measure glucose concentrations of one or more of 10 mM, 20 mM, 30 mM, 40 mM, and 50 mM.
43 . The biosensor of claim 42 , wherein the biosensor comprises a reaction layer comprising the recombinant FAD-conjugated glucose dehydrogenase.
44 . The biosensor of claim 43 , wherein the reaction layer further comprises a hydrophilic polymer and an electron acceptor.
45 . The biosensor of claim 44 , wherein the reaction layer is in contact with an electrode system comprising a working electrode, a counter electrode, and a reference electrode, each on an insulating base plate.
46 . The biosensor of claim 42 , which is capable of detecting glucose by detection of an oxidation current.
47 . The biosensor of claim 42 , which is capable of detecting glucose by a change in color intensity.
48 . The biosensor of claim 42 , which is capable of detecting glucose by a pH change.
49 . The biosensor of claim 42 , wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 5% or less with a value of enzymatic activity for D-glucose taken as 100%.
50 . The biosensor of claim 49 , wherein the recombinant FAD-conjugated glucose dehydrogenase has a value of enzymatic activity for maltose of 3% or less with a value of enzymatic activity for D-glucose taken as 100%.