Abstract: 【Aim】 Pyridoxal kinase (PLK) is a key enzyme related to VB₆ metabolism. In the previous study, the cDNA of PLK of the silkworm, Bombyx mori, was cloned, and several important and conservative amino acid residues were replaced in this protein by sequence alignment. This study aims to identify the effect of amino acid residues at specific position of PLK of B. mori on enzyme function. 【Methods】 The conserved sites Thr⁴⁷, Asn¹²¹, Ile⁵⁴, Arg⁸⁸ and Trp²³⁰ were site-mutated respectively by using over-lap extension. The expression plasmid pET-22b(+)-PLK was constructed and transformed to Escherichia coli Rosetta for induction and expression, and then the function of the recombinant protein was analyzed after expression product was purified using affinity chromatography. 【Results】 Compared with the wild type PLK, the PLK activities of the mutantions Thr⁴⁷, Ile⁵⁴ and Arg⁸⁸ were reduced by 82%, 58% and 85%, respectively, while the PLK activity of the mutantion Asn¹²¹ was hardly affected and the PLK activity of the mutantion Trp²³⁰ vanished. 【Conclusion】 In this study, the significance of selected amino acid residues of side chains on the catalytic function of PLK of B. mori was clarified.

Key words: Bombyx mori, pyridoxal kinase, site-directed mutation, expression, purification, functional identification, enzyme activity