Fructosyl amino acid oxidase

• US 8,497,083 B2
• Filed: 02/03/2012
• Issued: 07/30/2013
• Est. Priority Date: 08/03/2009
• Status: Active Grant

• Current Assignee: Roche Diabetes Care Inc. (Roche Holding AG)
• Original Assignee: Roche Diagnostics Operations Incorporated (Roche Holding AG), Ultizyme International Ltd.
• Inventors: Ikebukuro, Kazunori, Koji, Sode
• Primary Examiner(s): NASHED, NASHAAT T
• Application Number:
  US13/365,487
• Publication Number:
  US 20120208226A1
• Time in Patent Office: 543 Days
• Field of Search: None
• US Class Current: 435/25
• CPC Class Codes: C12N 9/0022 with oxygen as acceptor (1.4.3)
• CPC Class Codes: C12Q 1/005 involving specific analytes or enzymes including groups of enzymes, e.g. oxydases; C12Q1/004 takes precedence
• CPC Class Codes: G01N 33/6803 General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6806 Determination of free amino acids
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

In one form, a mutant fructosyl amino acid oxidase modified at an amino acid residue involved in a proton relay system is provided. The mutant fructosyl amino acid oxidase has reduced oxidase activity while substantially maintaining its dehydrogenase activity. Other forms include an assay device and assay method for measuring glycated protein. Still, other forms include unique methods, techniques, systems and devices involving a mutant fructosyl amino acid oxidase.

Claims

1. A mutant fructosyl amino acid oxidase, comprising a modification at a position corresponding to position 56 of the amino acid sequence set forth in SEQ ID NO:

1, wherein the modification includes a substitution of the amino acid residue Asn with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

2. The mutant fructosyl amino acid oxidase of claim 1, which includes a reduced oxidase activity as compared to the wild-type fructosyl amino acid oxidase, and an increased dehydrogenase activity compared to the wild-type fructosyl amino acid oxidase.

3. The mutant fructosyl amino acid oxidase of claim 1, which includes an oxidase activity of 30% or less of that of the wild-type fructosyl amino acid oxidase, and a dehydrogenase activity of 50% or more of the wild-type fructosyl amino acid oxidase.

4. The mutant fructosyl amino acid oxidase of claim 1, which includes an increased dehydrogenase activity compared to the wild-type fructosyl amino acid oxidase.

5. The mutant fructosyl amino acid oxidase of claim 1, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

1-12 wherein the amino acid residue Asn at a position corresponding to the position 56 of the amino acid sequence set forth in SEQ ID NO;

1 is substituted with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

6. The mutant fructosyl amino acid oxidase of claim 1, which includes the amino acid sequence set forth in SEQ ID NO:

1 wherein the amino acid residue Asn at the position 56 is substituted with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

7. The mutant fructosyl amino acid oxidase of claim 6, wherein the amino acid residues Ser Gly Tyr Gln Ala Leu Val Asp Ala Gly Leu Asp Ala Thr Asn Glu at positions 109-124 of SEQ ID NO:

1 are replaced with the sequence;

Lys Gln Tyr Gln Ala Leu His Asp Ala Gly Ala Gly Leu Glu Lys Thr His Ala of SEQ ID NO;

16.

8. The mutant fructosyl amino acid oxidase of claim 1, which includes the amino acid sequence set forth in SEQ ID NO:

2 wherein the amino acid residue Asn at position 47 is substituted with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

9. The mutant fructosyl amino acid oxidase of claim 1, which includes the amino acid sequence set forth in SEQ ID NO:

3 wherein the amino acid residue Asn at position 52 is substituted with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

10. The mutant fructosyl amino acid oxidase of claim 1, which includes the amino acid sequence set forth in SEQ ID NO:

7 wherein the amino acid residue Asn at the position 56 is substituted with an amino acid residue selected from Ala, Cys, Phe, Met and Val.

11. The mutant fructosyl amino acid oxidase of claim 10, wherein the amino acid residues Ile Arg Leu Arg Asn Lys Val Asp Leu Gln Met Ser at positions 61-72 of SEQ ID NO:

7 are replaced with the sequence Val Ser Leu Arg Asn Pro Val Asp Leu Gln Leu Ala of SEQ ID NO;

13.

14. A method for assaying a glycated protein in a sample, comprising contacting the sample with the fructosyl amino acid oxidase of claim 1, and measuring the amount of the glycated protein oxidized by the fructosyl amino acid oxidase.

15. A method for assaying HbAlc, comprising digesting HbAlc in a sample to generate fructosyl valine or fructosyl valyl histidine, contacting the fructosyl valine or fructosyl valyl histidine with the fructosyl amino acid oxidase of claim 1, and measuring the amount of oxidized fructosyl valine or fructosyl valyl histidine.

16. A device for assaying fructosyl valine, fructosyl valyl histidine, HbAlc or fructosyl hexapeptide in a sample, comprising the fructosyl amino acid oxidase of claim 1 and an electron transfer mediator.

17. A kit for assaying fructosyl valine, fructosyl valyl histidine, HbAlc or fructosyl hexapeptide in a sample, comprising the fructosyl amino acid oxidase of claim 1 and an electron transfer mediator.

18. An enzyme electrode, comprising the fructosyl amino acid oxidase of claim 1 which is immobilized on the electrode.

19. An enzyme sensor for assaying fructosyl valine, fructosyl valyl histidine, HbAlc or fructosyl hexapeptide, comprising the enzyme electrode of claim 18 as a working electrode.

12. A mutant fructosyl amino acid oxidase, comprising the amino acid sequence set forth in SEQ ID NO:

1 wherein the amino acid residues Val Ser Leu Arg Asn Pro Val Asp Leu Gln Leu Ala at positions 61-72 of SEQ ID NO;

1 are replaced with the sequence;

Ile Arg Leu Arg Asn Lys Val Asp Leu Gln Met Ser of SEQ ID NO;

14.

13. A mutant fructosyl amino acid oxidase, comprising the amino acid sequence set forth in SEQ ID NO:

1 wherein the amino acid residues Ser Gly Tyr Gln Ala Leu Val Asp Ala Gly Leu Asp Ala Thr Asn Glu at positions 109-124 of SEQ ID NO;

1 are replaced with the sequence;

Lys Gln Tyr Gln Ala Leu His Asp Ala Gly Ala Gly Leu Glu Lys Thr His Ala of SEQ ID NO;

16.