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【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】 To clone cyp107z gene from Streptomyces non-hygroscopicus ZB01 and purify it by heterologous expression in E. coli to determine the enzyme kinetic parameters of recombinant enzyme protein, which laid the foundation for further study of this gene. 【Method】 According to the conserved region of cyp gene, primers were designed to amplify the partial sequence of cyp107z gene in ZB01 genome. The full-length gene was obtained by chromosome walking technique. The prokaryotic expression vector was constructed by using pET28a expression vector and induced in E.coli. The recombinant protein was purified by Ni-NTA affinity chromatography. Avermectin was used as substrate to construct the recombinant protein in vitro catalytic system, and the enzyme kinetics parameters of the recombinant protein for avermectin reaction were calculated by measuring the consumption of NADPH in the system. 【Result】 A cyp107z homologous gene was amplified from the non-Streptomyces hygroscopicus ZB01 genome. The full-length gene was 1290 bp in length encoding a 429 amino acid residue named cyp107z13. The recombinant protein was induced and expressed in E. coli, and purified After the recombinase protein catalyzed avermectin Km value of 1.4μmol / L, Vmax 0.041μmol / min.mg, kcat 0.033 s-1. 【Conclusion】 The cyp107z13 gene was cloned from Streptomyces hygroscopicus ZB01. The heterologous expression of CYP107Z13 recombinant protein can catalyze the oxidation of abamectin as a substrate.