Thermostable mutants of pyrroloquinoline quinone dependent glucose dehydrogenase

• US 7,781,196 B2
• Filed: 10/14/2005
• Issued: 08/24/2010
• Est. Priority Date: 10/15/2004
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Schmuck, Rainer, Boenitz-Dulat, Mara, Kratzsch, Peter
• Primary Examiner(s): Pak; Yong D
• Application Number:
  US11/665,437
• Publication Number:
  US 20090305316A1
• Time in Patent Office: 1,775 Days
• Field of Search: None
• US Class Current: 435/190
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)

Abstract

The present invention relates to a mutant protein of PQQ-dependent s-GDH characterized in that in at least one of the positions 122 and 124 the amino acid lysine is present, wherein these positions correspond to the amino acid positions known from the A. calcoaceticus s-GDH wild-type sequence (SEQ ID NO: 2), it also discloses genes encoding such mutant s-GDH, and different applications of these s-GDH mutants, particularly for determining the concentration of glucose in a sample.

Claims

1. An isolated mutant protein of PQQ-dependent s-GDH, said mutant having at least 89% sequence identity with the A. calcoaceticus s-GDH wild-type sequence (SEQ ID NO:

2) and characterized in that in at least one of the positions 122 and 124 the amino acid lysine is present, wherein these positions correspond to the amino acid positions known from the A. calcoaceticus s-GDH wild-type sequence (SEQ ID NO;

     2).

2. The mutant of claim 1, wherein the amino acid at position 122 is a lysine.

3. The mutant of claim 1, wherein the amino acid at position 124 is a lysine.

4. The mutant of claim 1, wherein the amino acid lysine is present at both the positions 122 and 124.

5. The mutant according to claim 1 further comprising one or more amino acid substitutions, relative to the sequence of SEQ ID NO:

2, at one or more positions selected from the group consisting of positions 16, 22, 65, 76, 116, 120, 127, 143, 168, 169, 171, 177, 224, 227, 230, 231, 245, 246, 255, 277, 287, 294, 295, 299, 302, 305, 307, 308, 317, 321, 323, 341, 348, 349, 354, 355, 364, 378, 422, 425, 428 and 438.

6. The mutant according to claim 5, wherein said protein comprises an amino acid substitution at position 348 relative to the sequence of SEQ ID NO:

2.

7. The mutant according to claim 5, wherein said protein comprises an amino acid substitution at position 428 relative to the sequence of SEQ ID NO:

2.

8. The mutant according to claim 5, wherein said protein comprises an amino acid substitution at both positions 348 and 428.

9. The mutant of claim 6, wherein the native threonine at position 348 is replaced by alanine, glycine, or serine.

10. An isolated polynucleotide encoding the s-GDH mutant protein of claim 1.

11. An expression vector comprising an isolated polynucleotide as defined in claim 10 operably linked to a promoter sequence capable of promoting the expression of said polynucleotide in a host cell.

12. A host cell comprising the expression vector of claim 11.

13. A process for producing s-GDH variants comprising culturing the host cell of claim 12 under conditions suitable for production of the enzyme variants.

14. A method of detecting, determining or measuring glucose in a sample using an s-GDH mutant according to claim 1, said improvement comprising contacting the sample with the mutant.

15. The method of claim 14 further characterized in that said detection, determination or measurement of glucose is performed using a sensor or test strip device.

16. A device for the detection or measurement of glucose in a sample comprising an s-GDH mutant according to claim 1 and other reagents required for said measurement.

17. An isolated mutant protein of PQQ-dependent s-GDH comprising a sequence that differs from SEQ ID NO:

2 by 20 or less amino acid substitutions and/or insertions and includes an amino acid substitution at position 122, (relative to SEQ ID NO;

     2), and optionally one or more additional amino acid substitutions, relative to the sequence of SEQ ID NO;

2, at positions selected from the group consisting of positions 16, 22, 65, 76, 116, 120, 124, 127, 143, 168, 169, 171, 177, 224, 227, 230, 231, 245, 246, 255, 277, 287, 294, 295, 299, 302, 305, 307, 308, 317, 321, 323, 341, 348, 349, 354, 355, 364, 378, 422, 425, 426, 428, 430 and 438, and an optional insertion of proline at position 429, wherein at least one of positions 122 or 124 is substituted with the amino acid lysine, wherein said mutant has at least a two-fold improved substrate specificity for glucose as compared to one other selected sugar substrate and a thermo stability that is at least 20% of the thermo stability as measured for the wild-type enzyme.

18. The mutant protein according to claim 17 wherein the amino acid at position 428 is substituted with an amino acid selected from the group consisting of leucine, proline and valine.

19. The mutant protein according to claim 17 wherein the amino acid at position 348 is substituted with an amino acid selected from the group consisting of alanine, glycine and serine.

20. The mutant protein according to claim 17 wherein both amino acids at positions 122 and 124 are substituted with the amino acid lysine, the amino acid at position 348 is substituted with an amino acid selected from the group consisting of alanine, glycine and serine, and the amino acid at position 428 is substituted with an amino acid selected from the group consisting of leucine, proline and valine.

21. The mutant protein according to claim 17 wherein said protein differs from SEQ ID NO:

2 by amino acid substitutions at

a) positions 65, 122, 124, 171, 245, 341, 348, 426, 428, 430, and 436, or

b) positions 65, 122, 124, 171, 245, 341, 348, 426, 428, and 430 or

c) positions 122, 124, 171, 245, 246, 341, 348, 425, and 428.

22. The mutant protein according to claim 17 wherein said protein comprises a sequence that differs from SEQ ID NO:

2 by 20 or less amino acid substitutions and includes amino acid substitutions N122K, T348G and N428P (relative to SEQ ID NO;

     2), and optionally one or more additional amino acid substitutions, relative to the sequence of SEQ ID NO;

2, selected from the group consisting of Y171G, S124K, E245D, Q246H, M341, T425V, A426S and Q430M, and an optional insertion of proline at position 429, wherein said mutant has an at least two-fold improved substrate specificity for glucose as compared to at least one other selected sugar substrate and a thermo stability that is at least 20% of the thermo stability as measured for the wild-type enzyme.

23. The mutant protein according to claim 17 wherein said protein differs from SEQ ID NO:

2 only by the following amino acid substitutions N122K+S124K+Y171G+E245D+M341V+T348G+A426S+N428P+Q430M.