Genetically Engineered Pyrroloquinoline Quinone Dependent Glucose Dehydrogenase Comprising an Amino Acid Insertion

• US 20070243566A1
• Filed: 07/19/2005
• Published: 10/18/2007
• Est. Priority Date: 07/20/2004
• 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, Kratzsch, Peter, Boenitz-Dulat, Mara, Laggerbauer, Jessica
• Granted Patent: US 7,547,524 B2
• Time in Patent Office: Days
• US Class Current: 435/14
• 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) comprising an amino acid insertion between positions 428 and 429 as corresponding to the amino acid sequence known from Acinetobacter calcoaceticus, to genes encoding such variant s-GDH, to proteins of such s-GDH variants with improved substrate specificity for glucose, and to different applications of these s-GDH variants, particularly for determining concentrations of sugars, especially of glucose in a sample.

Claims

1. A variant of the soluble form of EC 1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH) said variant comprising at least one amino acid residue insertion between the amino acid positions corresponding to positions 428 and 429 of the s-GDH wild-type sequence known from A. calcoaceticus (SEQ ID NO:

2) and optionally in addition comprising one or more amino acid substitutions.

2. The variant according to claim 1 further characterized in that said inserted amino acid is selected from the group consisting of phenylalanine, leucine, methionine and proline.

3. The variant according to claim 1, wherein said inserted amino acid is proline.

4. The variant according to claim 1 further characterized in that it comprises an amino acid residue substitution at the amino acid position corresponding to position 348.

5. The variant according to claim 4 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.

6. The variant of PQQ-dependent s-GDH according to claim 1 further characterized in that it comprises at least one amino acid residue substitution at the amino acid position corresponding to position 428.

7. The variant of PQQ-dependent s-GDH according to claim 6 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.

8. A variant according to claim 1 comprising substitutions at both the positions 348 and 428.

9. A variant according to claim 1 additionally comprising a substitution at position 171.

10. A variant according to claim 1 additionally comprising a substitution at position 245.

11. A variant according to claim 1 additionally comprising a substitution at position 341.

12. A variant according to claim 1 additionally comprising a substitution at position 169.

13. A variant according to claim 1 additionally comprising a substitution at position 349.

14. An isolated polynucleotide encoding the s-GDH variant protein according to claim 1.

15. An expression vector comprising an isolated polynucleotide as defined in claim 14 operably linked to a promoter sequence capable of promoting the expression of said polynucleotide in a host cell.

16. A host cell comprising the expression vector of claim 15.

17. A process for producing s-GDH variants comprising culturing the host cell of claim 16 under conditions suitable for production of the enzyme variants.

18. An expression vector comprising an isolated polynucleotide as defined in claim 14 operably linked to a promoter sequence capable of promoting its expression in a cell-free peptide synthesis system.

19. A process for producing s-GDH variants with the construct of claim 18 in a cell-free peptide synthesis system under conditions suitable for production of the said enzyme variants.

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

21. The method of claim 20 further characterized in that said detection, determination or measurement of glucose is performed using a sensor or test strip device.

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