MODIFIED AMADORIASE AND METHOD FOR PRODUCING THE SAME, AGENT FOR IMPROVING SURFACTANT RESISTANCE OF AMADORIASE AND COMPOSITION FOR MEASURING HbA1c USING THE SAME

Mutation(s) for Improved Surfactant Resistance

[0294](1) Preparation of Recombinant Plasmid pKK223-3-CFP-T7 DNA

[0295]Escherichia coli strain JM109 (pKK223-3-CFP-T7) having a recombinant plasmid containing CFP-T7 gene (SEQ ID NO: 2) (see International Publication No. WO 2007 / 125779) was inoculated in 2.5 ml of LB-amp medium [1% (w / v) bactotrypton, 0.5% (w / v) peptone, 0.5% (w / v) NaCl, and 50 μg / ml ampicillin] and subjected to shake culture at 37° C. for 20 hours and a culture product was obtained. [0160]1

[0296]The culture product was centrifuged at 7,000 rpm for 5 minutes to collect strains. Then the recombinant plasmid pKK223-3-CFP-T7 was extracted and purified therefrom using the QIAGEN tip-100 kit (QIAGEN), and 2.5 μg DNA of the recombinant plasmid pKK223-3-CFP-T7 was obtained.

(2) Site-Directed Modification Operation of DNA of Recombinant Plasmid pKK223-3-CFP-T7

[0297]PCR was carried out under conditions described below using obtained DNA of the recombinant plasmid pKK223-3-CF

Accumulation of Mutation for Improved Surfactant Resistance

[0336]Based on the findings of mutations for enhancing surfactant resistance obtained in Example 1, multiple variants (a double variant, a triple variant, a quadruple variant, a quintuple variant, a sextuple variant, or a septuple variant) were tested to combine and accumulate these mutations in order to obtain an amadoriase having further increased surfactant resistance.

[0337]SEQ ID NO: 3 is the amino acid sequence of an amadoriase derived from the genus Coniochaeta into which a mutation for improving substrate specificity (E98A) and mutations for enhancing heat stability (F43Y, G184D, deletion of 3 carboxy-terminal amino acid residues) were introduced (hereinafter indicated with “CFP-D”), and is encoded by the gene of SEQ ID NO: 4. Mutations for enhancing surfactant resistance were accumulated using plasmid DNA in which CFP-D gene was inserted into pKK223-3 vector as a template. PCR reaction was carried out under the same con

Evaluation for Surfactant TTAC

[0347]Tetradecyltrimethylammonium chloride (hereinafter indicated with “TTAC”) was used in place of the surfactant CTAC used in Example 2 to evaluate the stability of CFP-D. The surfactant resistance of various modified amadoriases was evaluated in accordance with a measurement method for surfactant resistance according to Example 1, although under surfactant treatment conditions in which the amadoriases were each diluted in a 20 mM potassium phosphate buffer solution (pH 7.0) and mixed with 0.04% TTAC. The results are shown in Table 3-1. In this respect, it was confirmed that when the warmed sample was again measured for activity 30 minutes after 2-fold dilution in a BSA solution, there was no change in the activity value.

TABLE 3-1Plasmid asAmino AcidSEQ ID NO ofResidualPlasmidTemplateEnzymeMutationOligonucleotide UsedActivity (%)pKK223-3-NoneCFP-DNoneNone29.2CFP-DpKK223-3-pKK223-3-CFP-D1E44P23.2843.2CFP-D1CFP-DpKK223-3-pKK223-3-CFP-D2E44P / E340P16.1769.