Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase
First Claim
1. A mutant of the soluble form of EC 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said mutant characterized in that relative to the corresponding wild-type enzyme and with regard to at least one other selected sugar substrate, it has an at least two-fold increased substrate specificity for glucose.
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Abstract
The present invention relates to improved variants of soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenases (s-GDH), to genes encoding mutated s-GDH, to mutant proteins of s-GDH with improved substrate specificity for glucose, and to different applications of these s-GDH variants, particularly for determining concentrations of sugar, especially of glucose in a sample.
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Citations
30 Claims
- 1. A mutant of the soluble form of EC 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said mutant characterized in that relative to the corresponding wild-type enzyme and with regard to at least one other selected sugar substrate, it has an at least two-fold increased substrate specificity for glucose.
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6. A mutant of the soluble form of EC 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said mutant characterized in that
a) the substrate specific reactivity towards glucose is essentially equal to that of the wild-type enzyme, and b) the substrate specific reactivity towards maltose is 30% or less as compared to the wild-type enzyme.
- 9. A mutant protein of PQQ-dependent s-GDH comprising an amino acid residue substitution at the amino acid position corresponding to position 348 of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:
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10. A mutant protein of PQQ-dependent s-GDH comprising at least two amino acid residue substitutions at amino acid positions corresponding to positions of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:
- 24), said substituted amino acid positions being selected from the group consisting of positions 16, 22, 76, 116, 120, 127, 143, 168, 169, 171, 177, 227, 230, 231, 245, 255, 277, 295, 299, 308, 317, 341, 348, 349, 355, 422, 428 and 438, wherein the amino acid residue T348 is replaced.
- View Dependent Claims (11, 12, 14, 22, 23, 24, 25, 26, 27)
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13. A mutant protein of PQQ-dependent s-GDH comprising at least three amino acid residue substitutions at amino acid positions corresponding positions of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:
- 24), said substituted amino acid positions being selected from the group consisting of positions 171, 227, 230, 245, 341, 348, 349, and 428 wherein both the amino acid residues T348 and N428 are substituted.
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16. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of WPXaaVAPS (SEQ ID NO:
- 1), wherein said Xaa residue is an amino acid residue other than threonine.
- View Dependent Claims (17)
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18. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of TAGXaaVQK (SEQ ID NO:
- 2), wherein said Xaa residue is an amino acid residue other than asparagine.
- View Dependent Claims (19)
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20. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of ADGXaaNGL (SEQ ID NO:
- 3), wherein said Xaa residue is an amino acid residue other than glutamine.
- View Dependent Claims (21)
- 28. A method of detecting, determining or measuring glucose in a sample using a s-GDH mutant according to any of the preceeding claims, said improvement comprising contacting the sample with the mutant.
Specification