Abstract:

【Aim】 Pyridoxal kinase (PLK, EC 2.7.1.35) is a key enzyme related to vitamin B6 metabolism. In this study, recombinant pyridoxal kinase of Bombyx mori will be expressed, which will lay a foundation for further study on regulation mechanism, catalytic mechanism and structure of PLK in B. mori.【Methods】 After fusion expression vector harboring PLK gene of B. mori was constructed, the recombinant vector was transformed to Escherichia coli Rosetta for induction and expression, and then the characteristics of the purified recombinant protein was analyzed after the expression product was purified using affinity chromatography by Ni²⁺ column. 【Results】 Only one band was observed when the purified PLK was separated on SDS-PAGE. The PLK was enriched by 40fold and its specific activity was 1 800 U/mg. The optimum temperature and pH of this enzyme was about 50℃, and 5.5-6, respectively, and Zn²⁺ was the effective activator in enzymatic reaction. 【Conclusion】 The recombinant PLK exhibited the same catalytic characteristics as the native protein obtained from tissues of B. mori.

Key words: Bombyx mori, pyridoxal kinase, fusion expression, purification, enzyme activity