Inactivated enzyme variants and associated process and reagent system
First Claim
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1. A method for the determination of an analyte in a sample, comprising:
- (a) providing a reagent comprising a glucose dehydrogenase variant of SEQ ID NO;
6, wherein said variant differs from SEQ ID NO;
6 by an amino acid substitution at position 145 or 158, said variant having less than 3% residual enzymatic activity relative to the wild type enzyme of B. subtilis and retaining more than 20% of the coenzyme binding activity of the wild type enzyme of B. subtilis;
(b) contacting a sample suspected to contain an analyte with the reagent in the presence of a coenzyme, under conditions wherein the amount of the coenzyme bound to the glucose dehydrogenase variant to form a coenzyme glucose dehydrogenase variant complex is indicative of the amount of analyte to be determined; and
(c) detecting the coenzyme glucose dehydrogenase variant complex.
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Abstract
The present invention relates to oxidoreductase apoenzyme variants which are enzymatically inactive but have coenzyme-binding properties. Further, the present invention relates to DNA sequences encoding these oxidoreductase apoenzyme variants, expression vectors containing such DNA sequences and the use of these oxidoreductase apoenzyme variants in diagnostic applications.
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12 Claims
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1. A method for the determination of an analyte in a sample, comprising:
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(a) providing a reagent comprising a glucose dehydrogenase variant of SEQ ID NO;
6, wherein said variant differs from SEQ ID NO;
6 by an amino acid substitution at position 145 or 158, said variant having less than 3% residual enzymatic activity relative to the wild type enzyme of B. subtilis and retaining more than 20% of the coenzyme binding activity of the wild type enzyme of B. subtilis;
(b) contacting a sample suspected to contain an analyte with the reagent in the presence of a coenzyme, under conditions wherein the amount of the coenzyme bound to the glucose dehydrogenase variant to form a coenzyme glucose dehydrogenase variant complex is indicative of the amount of analyte to be determined; and (c) detecting the coenzyme glucose dehydrogenase variant complex. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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