Fad-conjugated glucose dehydrogenase gene

Abstract

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%.