Abstract: 【Aim】 To study the effects of single conserved amino acid residue of pyridoxine 5′-phosphate oxidase (PNPO) from Bombyx mori on its activity. 【Methods】 Lys¹¹¹(AAA) and Ser¹⁶⁰(AGC), two of the most conserved residues, were mutated to Glu (GAA) and Ala (GCC) by using over-lap extension， respectively. The obtained expression plasmids were over-expressed in Escherichia coli Rosetta by IPTG induction, and then the expressed products were purified with affinity chromatography and the activity of the purified PNPO were determined. 【Results】 The molecular mass of the target recombinant protein was ~45.0 kDa. Compared with the wild type PNPO, the activities of the mutants K111E and S160A were reduced by 78.0% and 67.4%, respectively. 【Conclusion】 The results suggest that the residues Lys¹¹¹ and Ser¹⁶⁰ are important to maintain the activity of PNPO. This study confirms the significance of single conserved amino acid residues on the catalytic function of PNPO of B. mori.

Key words: Bombyx mori, pyridoxine 5′-phosphate oxidase, site-directed mutation, gene expression, protein purification, enzyme activity