Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase

• US 20040005683A1
• Filed: 12/13/2002
• Published: 01/08/2004
• Est. Priority Date: 10/27/2000
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Knappe, Wolfgang-Reinhold, Schmuck, Rainer, Thym, Detlef, Shao, Zhixin, Beck, Daniela, Kratzsch, Peter
• Granted Patent: US 7,132,270 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 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said mutant characterized in that relative to the corresponding wild-type enzyme and with regard to at least one other selected sugar substrate, it has an at least two-fold increased substrate specificity for glucose.

2. The mutant according to claim 1 further characterized in that said selected sugar is selected from the group consisting of maltose and galactose.

3. The mutant according to claim 1 further characterized in that said selected sugar is maltose.

4. The mutant of PQQ-dependent s-GDH according to claim 1 further characterized in that said substrate specificity for glucose is improved at least 3-fold.

5. The mutant of PQQ-dependent s-GDH according to claim 1 further characterized in that said substrate specificity for glucose is improved at least 5-fold.

8. The mutant of a PQQ-dependent s-GDH according to claim 1 further characterized in that the wild-type s-GDH is isolated from a strain of the Acinetobacter species group consisting of A. calcoaceticus and A. baumannii.

30. A device for the detection or measurement of glucose in a sample comprising a s-GDH mutant according to any of claims 1-29 and other reagents required for said measurement.

6. A mutant of the soluble form of EC 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said mutant characterized in that

a) the substrate specific reactivity towards glucose is essentially equal to that of the wild-type enzyme, and

b) the substrate specific reactivity towards maltose is 30% or less as compared to the wild-type enzyme.

7. The mutant according to claim 6 further characterized in that said substrate specific reactivity towards maltose is 20% or less as compared to the wild-type enzyme.

9. A mutant protein of PQQ-dependent s-GDH comprising an amino acid residue substitution at the amino acid position corresponding to position 348 of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:

24).

15. The mutant according to claim 9 further characterized in that threonine at position 348 is substituted with an amino acid residue selected from the group consisting of alanine, glycine and serine.

10. A mutant protein of PQQ-dependent s-GDH comprising at least two amino acid residue substitutions at amino acid positions corresponding to positions of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:

24), said substituted amino acid positions being selected from the group consisting of positions 16, 22, 76, 116, 120, 127, 143, 168, 169, 171, 177, 227, 230, 231, 245, 255, 277, 295, 299, 308, 317, 341, 348, 349, 355, 422, 428 and 438, wherein the amino acid residue T348 is replaced.

11. The mutant of claim 10 further characterized in that at amino acid positions corresponding to positions of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:

24) the amino acid in position 348 and at least one of the following amino acid residues 16, 116, 120, 127, 169, 171, 177, 227, 255, 277, 299, 317, 355 and 438 are substituted.

12. The mutant protein of claim 10 comprising substitutions of the amino acid residues at positions 348 and 428.

14. The mutant according to claim 12 further characterized in that asparagine at position 428 is substituted with an amino acid residue selected from the group consisting of leucine, proline and valine.

22. An isolated polynucleotide encoding the s-GDH mutant protein according to any of claims 9 to 21.

23. An expression vector comprising an isolated polynucleotide as defined in claim 22 operably linked to a promoter sequence capable of promoting the expression of said polynucleotide in a host cell.

24. A host cell comprising the expression vector of claim 23.

25. A process for producing s-GDH variants comprising culturing the host cell of claim 24 under conditions suitable for production of the enzyme variants.

26. An expression vector comprising an isolated polynucleotide as defined in claim 22 operably linked to a promoter sequence capable of promoting its expression in a cell-free peptide synthesis system.

27. A process for producing s-GDH variants with the construct of claim 26 in a cell-free peptide synthesis system under conditions suitable for production of the said enzyme variants.

13. A mutant protein of PQQ-dependent s-GDH comprising at least three amino acid residue substitutions at amino acid positions corresponding positions of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:

24), said substituted amino acid positions being selected from the group consisting of positions 171, 227, 230, 245, 341, 348, 349, and 428 wherein both the amino acid residues T348 and N428 are substituted.

16. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of WPXaaVAPS (SEQ ID NO:

1), wherein said Xaa residue is an amino acid residue other than threonine.

17. The mutant protein of claim 16 further characterized in that said Xaa residue is glycine.

18. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of TAGXaaVQK (SEQ ID NO:

2), wherein said Xaa residue is an amino acid residue other than asparagine.

19. The mutant protein of claim 18 further characterized in that said Xaa residue is proline.

20. A mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of ADGXaaNGL (SEQ ID NO:

3), wherein said Xaa residue is an amino acid residue other than glutamine.

21. The mutant of claim 20 further characterized in that said Xaa residue is selected from the group consisting of aspartic acid, glutamic acid, methionine, proline, serine, alanine or glycine.

28. A method of detecting, determining or measuring glucose in a sample using a s-GDH mutant according to any of the preceeding claims, said improvement comprising contacting the sample with the mutant.

29. The method of claim 28 further characterized in that said detection, determination or measurement of glucose is performed using a sensor or test strip device.