Genetically engineered pyrroloquinoline quinone dependent glucose dehydrogenase comprising an amino acid insertion

• US 7,547,524 B2
• Filed: 07/19/2005
• Issued: 06/16/2009
• 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: Laggerbauer, Jessica, Knappe, Wolfgang-Reinhold, Schmuck, Rainer, Boenitz-Dulat, Mara, Kratzsch, Peter
• Primary Examiner(s): Saidha; Tekchand
• Application Number:
  US11/632,672
• Publication Number:
  US 20070243566A1
• Time in Patent Office: 1,428 Days
• Field of Search: 435/26, 435/189
• US Class Current: 435/26
• 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 PQQ-dependent soluble glucose dehydrogenase (s-GDH), said variant comprising the sequence of SEQ ID NO:

2 modified by the insertion of one amino acid residue 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), a substitution of threonine at position 348 of SEQ ID NO;

2 with an amino acid residue selected from the group consisting of alanine, glycine and serine, and optionally, one or more additional amino acid substitutions, wherein said variant is at least 90% identical to SEQ ID NO;

2 , and has s-GDH activity.

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 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 of SEQ ID NO:

2.

5. The variant of PQQ-dependent s-GDH according to claim 4 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.

6. A variant according to claim 1 comprising substitutions at both the positions corresponding to positions 348 and 428 of SEQ ID NO:

2.

7. A variant according to claim 1 additionally comprising a substitution at the amino acid position corresponding to position 171 of SEQ ID NO:

2.

8. A variant according to claim 1 additionally comprising a substitution at the amino acid position corresponding to position 245 of SEQ ID NO:

2.

9. A variant according to claim 1 additionally comprising a substitution at the amino acid position corresponding to position 341 of SEQ ID NO:

2.

10. A variant according to claim 1 additionally comprising a substitution at the amino acid position corresponding to position 169 of SEQ ID NO:

2.

11. A variant according to claim 1 additionally comprising a substitution at the amino acid position corresponding to position 349 of SEQ ID NO:

2.

12. 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.

13. The method of claim 12 further characterized in that said detection, determination or measurement of glucose is performed using a sensor or test strip device.