Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase

• US 7,547,535 B2
• Filed: 12/09/2005
• Issued: 06/16/2009
• Est. Priority Date: 10/27/2000
• Status: Expired due to Fees

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG)
• Inventors: Thym, Detlef, Beck, Daniela, Kratzsch, Peter, Knappe, Wolfgang-Reinhold, Schmuck, Rainer, Shao, Zhixin
• Primary Examiner(s): Saidha; Tekchand
• Application Number:
  US11/298,778
• Publication Number:
  US 20060148056A1
• Time in Patent Office: 1,285 Days
• Field of Search: 435/189, 435/200, 435/7.1, 435/287.1, 435/69.1, 435/320.1, 435/325, 435/190, 536/22.1, 536/23.2
• 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 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 pyrroloquinoline quinone (PQQ)-dependent soluble glucose dehydrogenase (s-GDH) mutant, comprising an amino acid sequence that is at least 90% identical to SEQ ID NO:

24 and wherein said mutant comprises an alanine or a serine substitution for threonine at position 348 of SEQ ID NO;

24 and wherein the mutant has s-GDH activity.

2. The mutant of claim 1, wherein at least one amino acid residue selected from the group consisting of Gln₇₆, Asp₁₄₃, Gln₁₆₈, Leu₁₆₉, and Asn₄₂₈, of SEQ ID NO:

24, is substituted with another amino acid.

3. The mutant of claim 1, wherein the glutamine at position 76 of SEQ ID NO:

24 is substituted with a different amino acid.

4. The mutant of claim 1 wherein the asparagine at position 428 of SEQ ID NO:

24 is substituted with another amino acid.

5. The mutant of claim 4, wherein at least one amino acid residue selected from the group consisting of Gln₇₆, Thr₁₂₇, and Asp₁₄₃, of SEQ ID NO:

24, is substituted with a different amino acid.

6. The mutant of claim 4, wherein at least one amino acid residue selected from the group consisting of Glu₂₄₅ and Met₃₄₁, of SEQ ID NO:

24, is substituted with a different amino acid.

7. The mutant of claim 3, wherein the glutamine at position 76, of SEQ ID NO:

24, is substituted with an amino acid selected from the group consisting of alanine, methionine, aspartic acid, proline, serine, glycine, and glutamic acid.

8. The mutant of claim 1 wherein amino acid residues other than glutamine and asparagine are present at positions 76 and 428, respectively, of SEQ ID NO:

24.

11. The mutant of claim 1 further wherein tyrosine, glycine, and serine residues are substituted at positions 227, 348, and 438, respectively, of SEQ ID NO:

24.

9. A PQQ-dependent s-GDH mutant, said mutant comprising an amino acid sequence at least 90% identical to SEQ ID NO:

24 wherein said mutant comprises a serine substitution for threonine at position 348 of SEQ ID NO;

24 and wherein the mutant has s-GDH activity.

10. A PQQ-dependent s-GDH mutant, said mutant comprising an amino acid sequence at least 90% identical to SEQ ID NO:

24 wherein said mutant comprises an alanine substitution for threonine at position 348 of SEQ ID NO;

24 and wherein the mutant has s-GDH activity.