Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase

• US 20060148056A1
• Filed: 12/09/2005
• Published: 07/06/2006
• Est. Priority Date: 10/27/2000
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Daniela Beck, Detlef Thym, Peter Kratzsch, Rainer Schmuck, Wolfgang-Reinhold Knappe, Zhixin Shao
• Inventors: Beck, Daniela, Kratzsch, Peter, Knappe, Wolfgang-Reinhold, Schmuck, Rainer, Thym, Detlef, Shao, Zhixin
• Granted Patent: US 7,547,535 B2
• Time in Patent Office: Days
• US Class Current: 435/189
• CPC Class Codes: C12N 9/0006 acting on CH-OH groups as donors (1.1)

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.

Claims

1. A mutant of the soluble form of EC 1.1.5.2, also known as pyrroloquinoline quinone (PQQ)-dependent soluble glucose dehydrogenase (s-GDH), having an amino acid residue other than threonine present at a position corresponding to position 348 of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

2. The mutant of claim 1, wherein the threonine at position 348 is substituted with an amino acid selected from the group consisting of alanine and serine.

3. The mutant of claim 1, wherein at least one amino acid residue selected from the group consisting of Gln₇₆, Asp₁₄₃, Gln₁₆₈, Leu₁₆₉, and Asn₄₂₈ is substituted with another amino acid.

4. The mutant of claim 1, wherein glutamine at position 76 is substituted with a different amino acid.

5. A mutant PQQ-dependent s-GDH having a leucine, proline, or valine residue in place of the asparagine present at a position corresponding to position 428 of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

6. The mutant of claim 5, wherein glutamine at position 76 is substituted with a different amino acid.

7. The mutant of claim 5, wherein the asparagine at position 428 is substituted with proline.

8. A mutant PQQ-dependent s-GDH having amino acid residues other than threonine and asparagine present at positions corresponding to positions 348 and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

9. The mutant of claim 8, wherein at least one amino acid residue selected from the group consisting of Gln₇₆, Thr₁₂₇, and Asp₁₄₃ is substituted with a different amino acid.

10. The mutant of claim 8, wherein at least one amino acid residue selected from the group consisting of Glu₂₄₅ and Met₃₄₁ is substituted with a different amino acid.

11. A mutant PQQ-dependent s-GDH having an amino acid residue other than glutamine present at a position corresponding to position 76 of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

12. The mutant of claim 11, wherein the glutamine at position 76 is substituted with an amino acid selected from the group consisting of alanine, methionine, aspartic acid, proline, serine, glycine, and glutamic acid.

13. A mutant PQQ-dependent s-GDH having amino acid residues other than glutamine, threonine, and asparagine present at positions corresponding to positions 76, 348 and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

14. A mutant PQQ-dependent s-GDH having a serine residue present at a position corresponding to position 348 of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

15. A mutant PQQ-dependent s-GDH having an alanine residue present at a position corresponding to position 348 of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

16. A mutant PQQ-dependent s-GDH having glycine, serine, histidine, and asparagine residues present at positions corresponding to positions 340, 348, 369, and 413, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

17. A mutant PQQ-dependent s-GDH having histidine, asparagine, and glycine residues present at positions corresponding to positions 171, 308, and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

18. A mutant PQQ-dependent s-GDH having aspartic acid, valine, and glycine residues present at positions corresponding to positions 171, 317, and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

19. A mutant PQQ-dependent s-GDH having aspartic acid, serine, arginine, histidine, and glycine residues present at positions corresponding to positions 16, 120, 177, 277, and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

20. A mutant PQQ-dependent s-GDH having threonine, threonine, arginine, and glycine residues present at positions corresponding to positions 116, 255, 299, and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

21. A mutant PQQ-dependent s-GDH having tyrosine, glycine, and serine residues present at positions corresponding to positions 227, 348, and 438, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

22. A mutant PQQ-dependent s-GDH having valine and glycine residues present at positions corresponding to positions 341 and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

23. A mutant PQQ-dependent s-GDH having glycine, glycine, and proline residues present at positions corresponding to positions 348, 349, and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

24. A mutant PQQ-dependent s-GDH having glycine, histidine, glycine, and proline residues present at positions corresponding to positions 171, 230, 348, and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

25. A mutant PQQ-dependent s-GDH having histidine, glycine, and proline residues present at positions corresponding to positions 230, 348, and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

26. A mutant PQQ-dependent s-GDH having phenylalanine, histidine, glycine, and proline residues present at positions corresponding to positions 227, 230, 348, and 428, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.

27. A mutant PQQ-dependent s-GDH having glutamic acid and glycine residues present at positions corresponding to positions 143 and 348, respectively, of the PQQ-dependent s-GDH wild-type sequence from A. calcoaceticus and having an amino acid sequence at least 90% identical thereto.