Amadoriase having improved specific activity

Patent metadata

Field	Value
Publication number	US-10934530-B2
Application number	US-201615563054-A
Country	US
Kind code	B2
Filing date	Apr 4, 2016
Priority date	Apr 3, 2015
Publication date	Mar 2, 2021
Grant date	Mar 2, 2021

Abstract

This invention provides an amadoriase having improved specific activity on a glycated substrate, compared with conventional amadoriase. Provided is an amadoriase comprising a substitution of the amino acid at the position corresponding to position 64 of the amino acid sequence as shown in SEQ ID NO: 1 with an amino acid selected from the group consisting of glycine, serine, methionine, leucine, threonine, valine, and isoleucine, a method for measurement of HbA1c, and a reagent kit for measurement of HbA1c using such amadoriase. Such method and kit for measurement enable rapid, simple, and accurate quantification of HbA1c.

First claim

The invention claimed is:

1. An amadoriase selected from the group consisting of the following (i) to
(i) an amadoriase comprising an amino acid sequence having 90% or higher sequence identity with the amino acid sequence as shown in SEQ ID NO: 1 over the full length, wherein the amino acid at the position corresponding to position 64 of the amino acid sequence as shown in SEQ ID NO: 1 is an amino acid selected from the group consisting of glycine, methionine, leucine, threonine, valine, and isoleucine and having amadoriase activity on a glycated peptide; and
(ii) an amadoriase comprising an amino acid sequence having 90% or higher sequence identity with the amino acid sequence as shown in SEQ ID NO: 1 over the full length, wherein the amino acid at the position corresponding to position 64 of the amino acid sequence as shown in SEQ ID NO: 1 is an amino acid selected from the group consisting of glycine, methionine, leucine, threonine, valine, and isoleucine and having amadoriase activity on α-fructosyl-valyl-histidyl-leucyl-threonyl-prolyl-glutamyl-glutamyl-lysine (αF8P).

2. The amadoriase according to claim 1 , comprising a substitution of 1 or more amino acid residues at a position corresponding to a position selected from the group consisting of (a) to (i) below:
(a) position 99 in SEQ ID NO: 1;
(b) position 62 in SEQ ID NO: 1;
(c) position 63 in SEQ ID NO: 1;
(d) position 102 in SEQ ID NO: 1;
(e) position 106 in SEQ ID NO: 1;
(f) position 110 in SEQ ID NO: 1;
(g) position 113 in SEQ ID NO: 1;
(h) position 355 in SEQ ID NO: 1; and
(i) position 419 in SEQ ID NO: 1.

3. The amadoriase according to claim 2 , wherein
(a) the amino acid at a position corresponding to position 99 in SEQ ID NO: 1 is serine;
(b) the amino acid at a position corresponding to position 62 in SEQ ID NO: 1 is alanine, aspartic acid, asparagine, glutamine, glutamic acid, glycine, valine, leucine, isoleucine, cysteine, serine, methionine, threonine, or proline;
(c) the amino acid at a position corresponding to position 63 in SEQ ID NO: 1 is alanine, histidine, or glycine;
(d) the amino acid at a position corresponding to position 102 in SEQ ID NO: 1 is lysine;
(e) the amino acid at a position corresponding to position 106 in SEQ ID NO: 1 is alanine, lysine, or arginine;
(f) the amino acid at a position corresponding to position 110 in SEQ ID NO: 1 is phenylalanine, histidine, leucine, or tyrosine;
(g) the amino acid at a position corresponding to position 113 in SEQ ID NO: 1 is lysine or arginine;
(h) the amino acid at a position corresponding to position 355 in SEQ ID NO: 1 is serine; and/or
(i) the amino acid at a position corresponding to position 419 in SEQ ID NO: 1 is lysine.

4. The amadoriase according to claim 1 , comprising a substitution of 1 or 2 amino acid residue(s) at a position corresponding to the position (j) or (k) below:
(j) position 68 in SEQ ID NO: 1; or
(k) position 356 in SEQ ID NO: 1.

5. The amadoriase according to claim 4 , wherein
(j) the amino acid at a position corresponding to position 68 in SEQ ID NO: 1 is asparagine; and/or
(k) the amino acid at a position corresponding to position 356 in SEQ ID NO: 1 is threonine.

6. The amadoriase according to claim 1 , comprising a substitution of 1 or more amino acid residues at a position corresponding to a position selected from the group consisting of (i) to (xiv) below:
(i) position 262;
(ii) position 257;
(iii) position 249;
(iv) position 253;
(v) position 337;
(vi) position 340;
(vii) position 232;
(viii) position 129;
(ix) position 132;
(x) position 133;
(xi) position 44;
(xii) position 256;
(xiii) position 231; and
(xiv) position 81.

7. The amadoriase according to claim 1 , which has specific activity on αF8P of 0.1 U/mg or greater.

8. The amadoriase according to claim 1 , which has specific activity on αF8P of 1 U/mg or greater.

9. The amadoriase according to claim 1 , which has specific activity on αF8P of 4 U/mg or greater.

10. The amadoriase according to claim 1 , which is derived from the genus Coniochaeta.

11. An amadoriase comprising the amino acid sequence as shown in SEQ ID NO: 211 or 213.

12. The amadoriase according to claim 1 , comprising a substitution of or more amino acid residue(s) at a position corresponding to a position selected from the group consisting of (l) to (q) below: 1:
(l) position 67 in SEQ ID NO: 1;
(m) position 72 in SEQ ID NO: 1;
(n) position 76 in SEQ ID NO: 1;
(o) position 96 in SEQ ID NO: 1;
(p) position 109 in SEQ ID NO: 1; and
(q) position 116 in SEQ ID NO: 1.

13. The amadoriase according to claim 12 , wherein
(l) the amino acid at a position corresponding to position 67 in SEQ ID NO: 1 is histidine;
(m) the amino acid at a position corresponding to position 72 in SEQ ID NO: 1 is serine;
(n) the amino acid at a position corresponding to position 76 in SEQ ID NO: 1 is alanine or phenylalanine;
(o) the amino acid at a position corresponding to position 96 in SEQ ID NO: 1 is glutamic acid;
(p) the amino acid at a position corresponding to position 109 in SEQ ID NO: 1 is arginine or lysine; and/or
(q) the amino acid at a position corresponding to position 116 in SEQ ID NO: 1 is arginine.

14. A reagent kit for measurement of hemoglobin A1c comprising the amadoriase according to claim 1 .

Assignees

• Kikkoman Corp

Inventors

• Ichiyanagi Atsushi
• KOMATSUZAKI Airi

Classifications

• C12Y105/03

  with oxygen as acceptor (1.5.3) · CPC title

• C12Q1/28

  involving peroxidase · CPC title

• C12Q1/26

  involving oxidoreductase · CPC title

• G01N33/72

  involving blood pigments, e.g. haemoglobin, bilirubin {or other porphyrins; involving occult blood} · CPC title

• G01N2333/805

  Haemoglobins; Myoglobins · CPC title