Modified glucose dehydrogenase

• US 6,103,509 A
• Filed: 09/04/1997
• Issued: 08/15/2000
• Est. Priority Date: 03/03/1997
• Status: Expired due to Fees

• Current Assignee: Koji Sode
• Original Assignee: Lifescan Incorporated (Duv Holding Corporation)
• Inventors: Sode, Koji
• Primary Examiner(s): Achutamurthy, Ponnathapu
• Assistant Examiner(s): Mayhew, Bradley S.
• Application Number:
  US08/923,109
• Time in Patent Office: 1,076 Days
• Field of Search: 435/14, 435/252.3, 435/190, 435/320.1, 536/23.2
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)

Abstract

The invention provides a modified pyrrolo-quinoline quinone glucose dehydrogenase (PQQGDH) enzyme protein which is modified to have a substrate specificity to glucose. The modified PQQGDH of the invention exhibits a high substrate specificity compared to the wild type PQQGDH, thus is useful in determining glucose concentration in clinical assays and food analyses. A gene coding for the modified PQQGDH and a glucose sensor comprising the modified PQQGDH are also disclosed.

Claims

1. A pyrrolo-quinoline quinone glucose dehydrogenase (PQQGDH) enzyme comprising the amino acid sequence Ala-Gly-Gly-Xaa-Gly-Ser-Phe (SEQ ID No:

11), wherein said Xaa residue is any amino acid residue other than histidine, wherein said SEQ ID No;

11 is found in a region corresponding to the sequence Ala-Gly-Gly-His-Gly-Ser-Phe (SEQ ID No;

10) found in wild-type E. coli PQQGDH, and wherein said PQQGDH enzyme has increased substrate specificity to glucose compared to that of said wild-type E. coli PQQGDH that comprises SEQ ID No;

10.

2. The enzyme claimed in claim 1 wherein said Xaa residue is selected from the group consisting of asparagine, aspartic acid, serine, tyrosine, lysine, leucine, tryptophane, glutamic acid and glutamine.

3. The enzyme claimed in claim 2 wherein said Xaa residue is aspartic acid or glutamic acid.

4. The enzyme claimed in claim 1 which is a chimeric protein.

5. An isolated polynucleotide encoding the PQQGDH enzyme of claim 1.

6. The polynucleotide claimed in claim 5 wherein said Xaa residue is selected from the group consisting of asparagine, aspartic acid, serine, tyrosine, lysine, leucine, tryptophane, glutamic acid and glutamine.

7. The polynucleotide claimed in claim 6 wherein said Xaa residue is aspartic acid or glutamic acid.

8. The polynucleotide claimed in claim 5 wherein said enzyme is a chimeric protein.

9. A plasmid containing the polynucleotide claimed in claim 5.

10. A transformant comprising the plasmid claimed in claim 9.

11. A kit for assaying glucose in a sample, the kit comprising the PQQGDH enzyme of claim 1 and suitable reagents.