摘要
【目的】克隆不吸水链霉菌ZB01中的cyp107z基因,在E.coli中异源表达纯化,测定重组酶蛋白的酶动力学参数,为该基因的进一步研究奠定基础。【方法】根据cyp基因保守区序列设计引物,扩增不吸水链霉菌ZB01基因组中cyp107z基因的部分序列,通过染色体步移技术获取全长基因。利用pET28a表达载体构建该基因原核表达载体并于E.coli中诱导表达,以Ni-NTA亲和层析纯化表达出的重组蛋白。以阿维菌素为底物,构建重组蛋白体外催化体系,通过测定体系中NADPH的消耗,计算重组蛋白催化阿维菌素反应的酶动力学参数。【结果】从不吸水链霉菌ZB01基因组扩增出一条cyp107z基因同源基因,全长1290 bp,编码429个氨基酸残基,命名为cyp107z13,在E.coli中诱导表达了该重组酶蛋白,纯化后的重组酶蛋白催化阿维菌素的Km值为1.4μmol/L,Vmax为0.041μmol/min.mg,kcat为0.033 s-1。【结论】从不吸水链霉菌ZB01中克隆到cyp107z13基因,异源表达的CYP107Z13重组蛋白能够催化以阿维菌素为底物的氧化反应。
[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-cyp107z13 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 μmol/L,the Vmax was 0.041 μmol /min.mg and the kcat was 0.033 s-1 in a reaction system with avermectin as substrate.[Conclusion] A cyp10z13 gene from Streptomyces ahygroscopicus ZB01 was cloned,the heterologous expressed recombinant protein can catalyze the oxidizing reaction with avermectin as substrate.
出处
《微生物学报》
CAS
CSCD
北大核心
2011年第3期410-416,共7页
Acta Microbiologica Sinica
基金
植物病虫害生物学国家重点实验室开放课题(2006PD5)~~
