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 - 34 . (canceled) 
     
     
         35 . A biosensor for measuring glucose in a sample liquid, comprising
 an electrode system and an enzyme reaction layer on an electrode of the electrode system,   the enzyme reaction layer comprising an electron acceptor and a flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD) obtained from  Aspergillus , wherein:   (a) the GLD has an activity toward glucose of catalyzing dehydrogenation of glucose in the presence of the electron acceptor and has an activity toward maltose of 5% or less with respect to the activity toward glucose;   (b) the GLD has an amino acid sequence comprising the sequences set forth in SEQ ID NOs. 8 to 12; and   (c) the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 10 μg or less per μg of protein.   
     
     
         36 . The biosensor of  claim 35 , wherein the electron acceptor is a ferricyanide compound. 
     
     
         37 . The biosensor of  claim 35 , wherein mannose is contained in the GLD. 
     
     
         38 . The biosensor of  claim 35 , wherein a N-type sugar chain or an O-type sugar chain is bound to the GLD. 
     
     
         39 . The biosensor of  claim 35 , wherein the biosensor can measure glucose concentrations of one or more of 10 mM, 20 mM, 30 mM and 40 mM. 
     
     
         40 . The biosensor of  claim 35 , wherein the total content of galactose, glucose, mannose, and arabinose contained in the GLD is 2 μg or less per μg of protein. 
     
     
         41 . The biosensor of  claim 35 , wherein the GLD is obtained from a mutant  Aspergillus.    
     
     
         42 . The biosensor of  claim 35 , wherein the GLD is obtained from a wild type  Aspergillus.    
     
     
         43 . The biosensor of  claim 35 , wherein the  Aspergillus  comprises  Aspergillus oryzae, Aspergillus japonicus , or  Aspergillus terreus.    
     
     
         44 . The biosensor of  claim 35 , wherein the GLD is recombinantly produced.