Glucose dehydrogenase
View Patent ↗A modified pyrroloquinoline quinone glucose dehydrogenase that exhibits a high selectivity for glucose is provided. A modified pyrroloquinoline quinone glucose dehydrogenase is disclosed in which the amino acid residue G at Position 99 of a pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) represented by SEQ ID NO: 1, or the amino acid residue G at Position 100 of the pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) represented by SEQ ID NO: 3, is substituted by the amino acid sequence TGZN (where Z is SX, S, or N and X is any amino acid residue). The modified PQQGDH of the present invention may additionally comprise one or more mutations selected from the group consisting of Q192G, Q192A, or Q192S; L193X; E277X; A318X; Y367A, Y367F, or Y367W; G451C; and N452X (where X is any amino acid residue).
1. A modified pyrroloquinoline quinone glucose dehydrogenase, wherein the amino acid residue G at Position 99 of a pyrroloquinoline quinone glucose dehydrogenase (PQQGDH) represented by SEQ ID NO: 1 is substituted by the amino acid sequence TGZN (where Z is SX, S, or N and X is any amino acid residue), and wherein from 1 to 10 of amino acid residues at Positions 1 to 98 or at Positions 100 to 478 of SEQ ID NO: 1, may be substituted by any other amino acid residue(s).
2. The modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 , wherein Z is SX (where X is any amino acid residue).
3. The modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 , wherein Z is SN.
4. The modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 , further comprising one or more mutations selected from the group consisting of the following amino acid substitutions:
Q192G, Q192A, or Q192S;
L193X (where X is any amino acid residue);
E277X (where X is any amino acid residue);
A318X (where X is any amino acid residue);
Y367A, Y367F, or Y367W;
G451C; and
N452X (where X is any amino acid residue).
5. The modified pyrroloquinoline quinone glucose dehydrogenase according to claim 4 , comprising any of the following combinations of amino acid mutations:
G99(TGSXN)+Q192G+L193E;
G99(TGSXN)+Q192S+N452P;
G99(TGSXN)+Q192G+L193E+N452P;
G99(TGSXN)+Q192S+N452P;
G99(TGSNN)+N452P;
G99(TGSNN)+Q192G+L193E+N452P;
G99(TGSNN)+Q192S+N452P;
G99(TGSNN)+Q192G+N452P;
G99(TGSNN)+L193E+N452P;
G99(TGSNN)+Q192S+L193M+N452P;
G99(TGSNN)+A318Y+N452P;
G99(TGSNN)+Q192G;
G99(TGSNN)+Q192S;
G99(TGSNN)+Q192A;
G99(TGSNN)+Q192G+L193E;
G99(TGSNN)+Q192S+L193X;
G99(TGSNN)+Q192S+L193M;
G99(TGSNN)+Q192S+L193T;
G99(TGSNN)+Q192S+E277X;
G99(TGSNN)+Q192S+N452X;
G99(TGSNN)+Q192S+L193X+A318Y+N452P;
G99(TGSNN)+Q192S+A318X+N452P;
G99(TGSNN)+Q192G+L193E+A318X+N452P;
G99(TGSKN)+Q192S+N452P;
G99(TGSRN)+Q192S+N452P;
G99(TGSWN)+Q192S+N452P;
G99(TGSN)+Q192S+N452P;
G99(TGSN)+Q192G+L193E;
G99(TGSN)+Q192S+L193M;
G99(TGNN)+Q192S+N452P;
G99(TGNN)+Q192G+L193E; or
G99(TGNN)+Q192S+L193M
where X is any amino acid residue.
6. The modified pyrroloquinoline quinone glucose dehydrogenase according to claim 5 , comprising any of the following combinations of amino acid mutations:
G99(TGSXN)+Q192G+L193E;
G99(TGSXN)+Q192S+N452P;
G99(TGSNN)+Q192G+L193E+N452P;
G99(TGSNN)+Q192S+L193M+N452P;
G99(TGSNN)+Q192G+L193E+A318K+N452P; or
G99(TGSNN)+Q192G+L193E+A318Q+N452P.
7. A method for analyzing glucose level using the modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 .
8. A glucose assay kit comprising the modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 .
9. An enzyme electrode comprising the modified pyrroloquinoline quinone glucose dehydrogenase according to claim 1 .
10. A glucose sensor comprising the enzyme electrode according to claim 9 as a working electrode.