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 an artificially produced flavin adenine dinucleotide (FAD)-linked glucose dehydrogenase (GLD), 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 recombinantly produced.