Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same

Abstract

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.

1 - 37 . (canceled) 
     
     
         38 . A method for producing a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) having a glucose dehydrogenation activity, comprising:
 cultivating a transformed host cell comprising a polynucleotide encoding the GLD, expressing the GLD, and collecting the GLD,
 wherein the GLD catalyzes dehydrogenation of glucose in presence of an electron acceptor and has an activity toward maltose of 5% or less with respect to an activity toward glucose, and the GLD has an amino acid sequence comprising sequences of the coenzyme-linked glucose dehydrogenase set forth in SEQ ID NOs. 8 to 12, 
 wherein the polynucleotide is not cDNA. 
   
     
     
         39 . The method according to  claim 38 , wherein the GLD has a subunit molecular weight of approximately 63 kDa, wherein the term “subunit molecular weight” refers to a subunit molecular weight determined by subjecting the GLD without sugar chains to polyacrylamide gel electrophoresis (SDS-PAGE). 
     
     
         40 . The method according to  claim 39 , wherein the GLD catalyzes a reaction in which a hydroxyl group at a 1-position of glucose is oxidized, and the glucose is converted to glucono-δ-lactone. 
     
     
         41 . The method according to  claim 40 , wherein the GLD catalyzes dehydrogenation of glucose in presence of an electron acceptor. 
     
     
         42 . A method for producing a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) having a glucose dehydrogenation activity, comprising:
 cultivating a transformed host cell comprising a polynucleotide encoding the GLD, expressing the GLD, and collecting the GLD,
 wherein the polynucleotide encoding the GLD comprises the sequences set forth in SEQ ID NOs. 5 to 7, and the GLD has the following properties (a) to (d): 
 (a) having a subunit molecular weight of approximately 63 kDa, wherein the term “subunit molecular weight” refers to a subunit molecular weight determined by subjecting the GLD without sugar chains to polyacrylamide gel electrophoresis (SDS-PAGE); 
 (b) utilizing a FAD as a coenzyme; 
 (c) catalyzing a reaction in which a hydroxyl group at a 1-position of glucose is oxidized, and the glucose is converted to glucono-δ-lactone; and 
 (d) having an activity toward maltose of 5% or less with respect to an activity toward glucose, wherein the polynucleotide is not cDNA. 
   
     
     
         43 . A method for producing a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) having a glucose dehydrogenation activity, comprising:
 cultivating a transformed host cell comprising a polynucleotide encoding the GLD, expressing the GLD, and collecting the GLD,
 wherein the GLD catalyzes dehydrogenation of glucose in presence of an electron acceptor and has an activity toward maltose of 5% or less with respect to an activity toward glucose, and the GLD includes: 
 (a) an amino acid sequence set forth in SEQ ID NO: 2 or an amino acid sequence set forth in amino acids 20 to 592 of SEQ ID NO: 2; or 
 (b) an amino acid sequence with a homology of at least 90% to the amino acid sequence set forth in SEQ ID NO: 2 or the amino acid sequence set forth in amino acids 20 to 592 of SEQ ID NO: 2 
 wherein the polynucleotide is not cDNA. 
   
     
     
         44 . The method according to  claim 38 , wherein the GLD is derived from a microorganism of an  Aspergillus  genus,  Penicillium  genus, or  Ganoderma  genus. 
     
     
         45 . The method according to  claim 42 , wherein the GLD is derived from a microorganism of an  Aspergillus  genus,  Penicillium  genus, or  Ganoderma  genus. 
     
     
         46 . The method according to  claim 43 , wherein the GLD is derived from a microorganism of an  Aspergillus  genus,  Penicillium  genus, or  Ganoderma  genus. 
     
     
         47 . The method according to  claim 44 , wherein the GLD is derived from a microorganism of an  Aspergillus terrous  ( A terrous ). 
     
     
         48 . The method according to  claim 45 , wherein the GLD is derived from a microorganism of an  Aspergillus terreus  ( A terreus ). 
     
     
         49 . The method according to  claim 46 , wherein the GLD is derived from a microorganism of an  Aspergillus terrous  ( A terreus ).