Weide Liu1, Xiliang Jiang, Ying Ji, Jing Lniu, Mei Li. 1. Key Laboratory for Biological Control of Ministry of Agriculture, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100081 China.
Abstract
OBJECTIVE: We cloned and expressed a cytochrome P450 gene cyp107z from Streptomyces ahygroscopicus ZB01, and determined the kinetic parameters of the recombinant enzyme in vitro. METHOD: Degenerate primers were designed by the conserved sequence of cyp genes and were used to amplify partial sequence of cyp107z gene from Streptomyces ahygroscopicus ZB01 genome. The full-length cyp107z gene sequence was obtained by genome walking, and linked with pET28a to construct pET-cyp1O7z13 expressing vector which was then transferred into Escherichia coli, and the expressed recombinant protein was purified by Ni-NTA affinity chromatography. The catalysis system of the recombinant protein was constructed with avermectin as substrate, and the kinetic parameters of the recombinant protein were determined by monitoring the consumption of NADPH in the system in vitro. RESULTS: A cyp107z homologous gene named cyp107z13 was cloned from Streptomyces ahygroscopicus ZB01 genome, which was 1290 bp in length encoding 429 amino acid residues. The Km of purified recombinant protein of CYP107Z13 expressed in E. coli was 1.4 micromol/L, the Vmax was 0.041 micromol/min x mg and the k(cat), was 0.033 s(-1) in a reaction system with avermectin as substrate. CONCLUSION: A cyp10z3 gene from Streptomyces ahygroscopicus ZB01 was cloned, the heterologous expressed recombinant protein can catalyze the oxidizing reaction with avermectin as substrate.
OBJECTIVE: We cloned and expressed a cytochrome P450 gene cyp107z from Streptomyces ahygroscopicus ZB01, and determined the kinetic parameters of the recombinant enzyme in vitro. METHOD: Degenerate primers were designed by the conserved sequence of cyp genes and were used to amplify partial sequence of cyp107z gene from Streptomyces ahygroscopicus ZB01 genome. The full-length cyp107z gene sequence was obtained by genome walking, and linked with pET28a to construct pET-cyp1O7z13 expressing vector which was then transferred into Escherichia coli, and the expressed recombinant protein was purified by Ni-NTA affinity chromatography. The catalysis system of the recombinant protein was constructed with avermectin as substrate, and the kinetic parameters of the recombinant protein were determined by monitoring the consumption of NADPH in the system in vitro. RESULTS: A cyp107z homologous gene named cyp107z13 was cloned from Streptomyces ahygroscopicus ZB01 genome, which was 1290 bp in length encoding 429 amino acid residues. The Km of purified recombinant protein of CYP107Z13 expressed in E. coli was 1.4 micromol/L, the Vmax was 0.041 micromol/min x mg and the k(cat), was 0.033 s(-1) in a reaction system with avermectin as substrate. CONCLUSION: A cyp10z3 gene from Streptomyces ahygroscopicus ZB01 was cloned, the heterologous expressed recombinant protein can catalyze the oxidizing reaction with avermectin as substrate.