Cholesterol oxidase mutants, compositions, devices, kits and uses thereof
AssigneesUltizymer International, Ltd., Roche Diabetes Care, Inc.
InventorsKatsuhiro Kojima, Kazushige Mori, Sode Koji
Life of this patent
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Abstract
Compositions, devices, kits and methods are disclosed for assaying cholesterol with a cholesterol oxidase mutant that has been modified at an amino acid residue involved in the active site. The cholesterol oxidase mutant has reduced oxidase activity while substantially maintaining its dehydrogenase activity.
Claims15
A cholesterol oxidase mutant modified at one or more amino acid positions selected from:
- (a). a position corresponding to position 159 of SEQ ID NO:
- 1 by substituting the amino acid residue Met with an amino acid residue selected from the group consisting of Phe, Leu, Val, Cys, Ile, Ala, Gln, Tyr, Lys, and Ser or by substituting the amino acid residue Ile with an amino acid residue selected from the group consisting of Phe, Leu, Val, Cys, Ala, Gln, Tyr, Lys and Ser;
- (b). a position corresponding to position 228 of SEQ ID NO:
- 1 by substituting the amino acid residue Val, Met or Ile with an amino acid residue selected from the group consisting of Ala, Thr, Lys, Cys, Ser, Gly, Glu, Tyr, Pro, Asn, Gln, Trp and His;
- and
- (c). a position corresponding to position 396 of SEQ ID NO:
- 1 by substituting the amino acid residue Phe with an amino acid residue selected from the group consisting of Trp, Ser, Thr, Lys, Ala, Asn, His and Asp,
- wherein the cholesterol oxidase mutant has at least about 95% sequence identity to any one of SEQ ID NOS:
- 1-5.
A cholesterol oxidase mutant comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:
- 1-5 modified at one or more positions corresponding to position 159, 228 or 369 of SEQ ID NO:
- 1, wherein the cholesterol oxidase mutant has a reduced oxidase activity when compared to a wild-type cholesterol oxidase.
A method of assaying cholesterol in a sample, the method comprising the steps of:
- contacting the sample with a cholesterol oxidase mutant modified at one or more amino acid positions selected from:
- (a). a position corresponding to position 159 of SEQ ID NO:
- 1 by substituting the amino acid residue Met with an amino acid residue selected from the group consisting of Phe, Leu, Val, Cys, Ile, Ala, Gln, Tyr, Lys, and Ser or by substituting the amino acid residue Ile with an amino acid residue selected from the group consisting of Phe, Leu, Val, Cys, Ala, Gln, Tyr, Lys and Ser,
- (b). a position corresponding to position 228 of SEQ ID NO:
- 1 by substituting the amino acid residue Val, Met or Ile with an amino acid residue selected from the group consisting of Ala, Thr, Lys, Cys, Ser, Gly, Glu, Tyr, Pro, Asn, Gln, Trp and His, and (c). a position corresponding to position 396 of SEQ ID NO:
- 1 by substituting the amino acid residue Phe with an amino acid residue selected from the group consisting of Trp, Ser, Thr, Lys, Ala, Asn, His and Asp;
- and
- measuring an amount of the cholesterol oxidized by the cholesterol oxidase,
- wherein the cholesterol oxidase mutant has at least 95% sequence identity to any one of SEQ ID NOS:
- 1-5.
Dependencies are inferred from the claim text.