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US 7,803,592 B2Granted
Mutant glucose dehydrogenase
AssigneeARKRAY, Inc.
InventorHideaki Yamaoka
Life of this patent
Estimating the term…
Abstract
Substrate specificity for glucose of a glucose dehydrogenase having the amino acid sequence of SEQ ID NO: 13 is improved by substituting another amino acid residue for the amino acid residue at position 472 and/or 475.
Claims14
An isolated mutant glucose dehydrogenase exhibiting improved substrate specificity to glucose, which is
- (1) a mutant of the protein comprising the amino acid sequence of SEQ ID NO:
- 13, wherein said mutant consists of amino acid substitution(s) at positions 472 and/or 475 as listed below in (A)-(C);
- or
- (2) a mutant of the protein comprising the amino acid sequence of SEQ ID NO:
- 13 wherein said mutant consists of amino acid substitution(s) at positions 472 and/or 475 as listed below in (A)-(C) and consists of substitution, deletion, insertion or addition of one to ten amino acid residues at position(s) other than the positions listed below and wherein mutants (1) or (2) have glucose dehydrogenase activity (numerals represent a position in the amino acid sequence, the amino acid residues represent an amino acid residue after substitution at the position, and “+” means that two amino acid substitutions are simultaneously included), wherein improved substrate specificity for mutants (1) or (2) is a decrease of at least 10% in the ratio of reactivity to maltose/reactivity to glucose compared to the glucose dehydrogenase of SEQ ID NO:
- 13:
- (A) 472Arg, 472Asn, 472Asp, 472Cys, 472Glu, 472Gly, 472His, 472lle, 472Leu, 472Met, 472Phe, 472Pro, 472Ser, 472Trp, 472Tyr, or 472Val;
- (B) 475Asp, 475Cys, 475Glu, 475Gly, 475His, 475Met, 475Phe, 475Ser, 475Tyr, or 475Val;
- or
- (C) 472Arg +475(Asp, Glu, Gly, His, Phe, Ser, Tyr),
- 472Asn +475(Asp, Gly, His, Phe, Ser, Tyr),
- 472Asp +475(His, Phe, Ser, Val),
- 472Cys +475(Asp, Gly, His, Phe, Ser),
- 472Glu +475(Asp, Glu, Gly, His, Phe, Ser, Tyr),
- 472Gly +475(Asp, Cys, Gly, Met, Phe, Ser, Tyr),
- 472His +475(Cys, Glu, His, Met, Phe, Ser, Tyr),
- 472lle +475(Asp, Cys, Glu, Gly, His, Met, Phe, Ser, Tyr),
- 472Leu +475(Asp, Gly, His, Phe, Ser, Tyr),
- 472Met +475(Asp, Gly, His, Phe, Ser),
- 472Phe +475(Asp, Glu, Gly, His, Met, Phe, Ser, Tyr),
- 472Pro +475His
- 472Ser +475(Asp, Glu, Gly, His, Phe, Ser),
- 472Trp +475(His, Phe, Ser),
- 472Tyr +475(Asp, His, Phe, Ser), or
- 472Val +475(Asp, Glu, Gly, His, Phe, Ser).
22 more elements
An isolated glucose dehydrogenase, which is
- (1) a mutant of the protein comprising the amino acid sequence of SEQ ID NO:
- 13, wherein said mutant consists of amino acid substitution(s) at positions 472 and/or 475, or
- (2) a mutant of the protein comprising the amino acid sequence of SEQ ID NO:
- 13, wherein said mutant consists of amino acid substitution(s) at positions 472 and/or 475 and consists of substitution, deletion, insertion or addition of one to ten amino acid residues at position other than positions 472 and/or 475, wherein mutants 1 or 2 have a glucose dehydrogenase activity, and wherein:
- (i) the substitutions at positions 472 and/or 475 in mutants (1) or (2) consist of replacement of at least either the arginine residue at position 472 or the asparagine residue at position 475 in the amino acid sequence of SEQ ID NO:
- 13 with another amino acid residue, and
- (ii) a ratio of specific activity for glucose and specific activity for maltose ((reactivity to maltose/reactivity to glucose)×100) of the glucose dehydrogenase of mutants (1) or (2) is reduced by 10% or more compared with that of the glucose dehydrogenase of SEQ ID NO:
- 13.
5 more elements
Dependencies are inferred from the claim text.