NOVEL PROTEIN HAVING FRUCTOSYL VALYL HISTIDINE OXIDASE ACTIVITY, MODIFIED PROTEIN, AND USE OF THE PROTEIN OR THE MODIFIED PROTEIN

• US 20110195444A1
• Filed: 10/06/2009
• Published: 08/11/2011
• Est. Priority Date: 10/10/2008
• Status: Active Grant

• Current Assignee: Toyobo Company Limited
• Original Assignee: Gekkeikan Sake Co., Ltd., Toyoda Gosei Company Limited (Toyota Motor Corporation)
• Inventors: Nishiya, Yoshiaki, Hirao, Rie, Kitabayashi, Masao, Hata, Yoji, Ishida, Hiroki
• Granted Patent: US 8,993,255 B2
• Time in Patent Office: Days
• US Class Current: 435/25
• CPC Class Codes: C12N 9/0022 with oxygen as acceptor (1.4.3)
• CPC Class Codes: C12Q 1/26 involving oxidoreductase
• CPC Class Codes: G01N 33/723 Glycosylated haemoglobin

Abstract

The present invention provides: a protein having a fructosyl amino acid oxidase activity which protein is useful for measurement of a glycosylated protein (particularly, glycosylated hemoglobin); a modified protein thereof; and use of the protein or the modified protein. The protein of the present invention is, for example, a fructosyl valyl histidine oxidase derived from Phaeosphaeria nodorum, the fructosyl valyl histidine oxidase having excellent thermal stability and substrate specificity and also having a small Km value to fructosyl valyl histidine. This allows a glycosylated protein measuring reagent to be stored in a long time and measurement accuracy of the glycosylated protein measuring reagent to be improved.

Claims

1. A protein having a fructosyl valyl histidine oxidase activity, the protein being any one of proteins (I) through (III), below:

(I) a protein comprising the amino acid sequence shown in SEQ ID NO;

1;

(II) a protein comprising the amino acid sequence shown in SEQ ID NO;

1 in which one or a few amino acid residues are replaced, deleted, inserted and/or added, the protein having the fructosyl valyl histidine oxidase activity; and

(III) a protein comprising an amino acid sequence having 86.0% or greater of homology with the amino acid sequence shown in SEQ ID NO;

1, the protein having the fructosyl valyl histidine oxidase activity.

2. The protein as set forth in claim 1, wherein:

the protein is the protein described in (II) or (III), and isoleucine at 58th position from an amino terminal and/or glycine at 110th position from the amino terminal are/is replaced with another amino acid.

3. The protein as set forth in claim 2, wherein:

the protein is the protein described in (II) or (III), and phenylalanine at 282th position from the amino terminal is replaced with another amino acid.

4. The protein as set forth in claim 2, wherein:

the isoleucine at 58th position from the amino terminal is replaced with methionine, threonine, alanine, asparagine, serine, valine or leucine.

5. The protein as set forth in claim 2, wherein:

the glycine at 110th position from the amino terminal is replaced with glutamine, methionine, glutamic acid, threonine, alanine, cysteine, histidine, lysine, asparagine, arginine, serine, valine, leucine, asparagine acid, isoleucine, tyrosine or phenylalanine.

6. The protein as set forth in claim 3, wherein:

the phenylalanine at 282th position from the amino terminal is replaced with tyrosine.

7. A polynucleotide that encodes for a protein having a fructosyl valyl histidine oxidase activity, the polynucleotide being any one of polynucleotides (IV) through (VII), below:

(IV) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO;

2;

(V) a polynucleotide comprising the nucleotide sequence shown in SEQ ID NO;

2 in which 1 through 30 nucleotides are replaced, deleted, inserted and/or added, the polynucleotide encoding for the protein having the fructosyl valyl histidine oxidase activity;

(VI) a polynucleotide that encodes for a protein as set forth in claim 1; and

(VII) a polynucleotide that hybridizes, under a stringent condition, with a polynucleotide comprising a nucleotide sequence complementary to that of any one of the polynucleotides (IV) through (VI), the polynucleotide encoding for the protein having the fructosyl valyl histidine oxidase activity.

8. A recombinant vector including a polynucleotide as set forth in claim 7.

9. A transformant transformed from a host by used of a recombinant vector as set forth in claim 8.

10. A method for producing a protein having a fructosyl valyl histidine oxidase activity, comprising the steps of:

culturing a transformant as set forth in claim 9 so that the protein having the fructosyl valyl histidine oxidase activity is produced; and

collecting the protein.

11. A method for measuring a glycosylated protein, comprising the step of causing a protein as set forth in claim 1 to act on a glycosylated amine.

12. A method for measuring glycosylated hemoglobin included in a sample, comprising the steps of:

preparing a glycosylated amine by use of the glycosylated hemoglobin included in the sample; and

causing a protein as set forth in claim 1 to act on the sample.

13. A kit for measuring a glycosylated protein, the kit including a protein as set forth in claim 1.

14. A sensor for measuring a glycosylated protein, the sensor including a protein as set forth in claim 1.