Glycated hemoglobin oxidase variant and method for measurement

A method for producing glycated hemoglobin oxidase by modifying an amadoriase or A1c oxidase comprising the following steps:

• (i) obtaining an amadoriase gene or A1c oxidase gene;
• (ii) integrating the amadoriase gene or A1c oxidase gene into a vector, transforming the host cell, expressing the amadoriase or A1c oxidase, and isolating the expressed product;
• (iii) measuring the relative activity (HbS1c/HbA1c) and/or the relative activity (HbC1c/HbA1c) of the expressed product;
• (iv) modifying the amadoriase gene or A1c oxidase gene such that, when the amino acid sequence of the amadoriase or A1c oxidase is aligned with the amino acid sequence of SEQ ID NO:
• 1, the amino acid at the position corresponding to position 113, 109, 106, or 102 of the amino acid sequence of SEQ ID NO:
• 1 is modified into an amino acid selected from the group consisting of glutamic acid, aspartic acid, alanine, serine, threonine, asparagine, glutamine, cysteine, glycine, proline, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine, and tryptophan;
• (v) integrating the modified gene into a vector, transforming a host cell, expressing the modified amadoriase or A1c oxidase, and isolating the expressed product;
• (vi) measuring the relative activity (HbS1c/HbA1c) and/or the relative activity (HbC1c/HbA1c) of the expressed product of the modified amadoriase or A1c oxidase and comparing the measured values with the values measured in step (iii);
• (vii) when the relative activity (HbS1/HbA1c) of the modified amadoriase or A1c oxidase is 1.1 times or greater than the relative activity (HbS1c/HbA1c) of the amadoriase or A1c oxidase before modification and/or the relative activity (HbC1c/HbA1c) of the modified amadoriase or A1c oxidase is 1.1 times or greater than the relative activity (HbC1c/HbA1c) of the amadoriase or A1c oxidase before modification, designating the modified amadoriase or A1c oxidase as a glycated hemoglobin oxidase;
• and
• (viii) repeating steps (iv) to (vi) on the glycated hemoglobin oxidase of step (vii), according to need.