阴沟肠杆菌WJ-1中甲氰菊酯降解酶基因pytZ的克隆、表达和重组酶特性研究

Cloning,Expression and Characterization of Fenpropathrin Degrading Enzyme Gene pytZ from Enterobacter cloacae WJ-1

【目的】农药降解酶通常比农药降解菌更能耐受异常环境,具有更宽广的降解谱。为获得高产率和高质量的农药降解酶,在实验室前期获得阴沟肠杆菌WJ-1和证实具有农药降解酶基因pytZ的基础上,本研究拟将阴沟肠杆菌WJ-1农药降解酶基因pytZ重组表达于受体菌Escherichia coli BL21。并对重组菌农药降解酶的生理生化特性进行深入研究。【方法】通过PCR技术扩增获得阴沟肠杆菌WJ-1农药降解酶基因pytZ,构建pMD19重组质粒,再与pET-24a(K+)表达载体进行连接,构建基因工程菌E.coli BL21/pET-24a(K+)-pytZ。将重组菌进行摇瓶培养。使用不同温度、pH值、IPTG浓度探讨重组酶酶促反应动力学参数。【结果】获取得到基因工程菌E.coli BL21/pET-24a(K+)-pytZ。30℃和IPTG诱导浓度为0.8 mmol/L时,摇瓶培养48 h后可以获得最高的农药降解酶产量,酶活力达到15.3 U/mL。重组菌农药降解酶的酶促反应最优温度为35℃,25~45℃热稳定良好;重组菌农药降解酶的酶促反应最优pH值为8.0,pH 7.0~9.0酸碱稳定良好;重组菌农药降解酶对底物甲氰菊酯的反应动力学参数,米氏常数K_(m)为0.076 mmol/L,最大反应速率V_(max)为3.023 mmol/(L·min)。【结论】利用基因工程菌Escherichia coli BL21/pET-24a(K+)-pytZ生产得到更大量的甲氰菊酯降解酶,纯度更高,具有更好的温度和pH值适应性,反应动力学参数显示其降解效率更高。

【Objective】The pesticide degrading enzymes were generally more tolerant to abnormal environment and had wider degradation spectrum than pesticide degrading bacteria.In order to obtain high yield and high quality pesticide degrading enzyme,based on the previous acquisition of Enterobacter cloacae WJ-1 and the confirmation of its pesticide degrading enzyme gene pytZ in the laboratory,the pesticide degrading enzyme gene pytZ of E.cloacae WJ-1 was recombined and expressed in the recipient strain Escherichia coli BL21.And the physiological and biochemical characteristics of the recombinant enzyme were studied.【Method】The pesticide degrading enzyme gene pytZ of E.cloacae WJ-1 was amplified by PCR,and the recombinant plasmid pMD19 was constructed.Then the recombinant plasmid pMD19 was ligated with pET-24 a(K+)expression vector to construct the genetically engineered strain Escherichia coli BL21/pET-24 a(K+)-pytZ.The recombinant bacteria were cultured in shake flask.The kinetic parameters of recombinant enzyme were studied by using different temperature,pH value and IPTG concentration.【Result】The E.coli BL21/pET-24 a(K+)-pytZ was obtained.At 30℃ and IPTG induction concentration of 0.8 mmol/L,the highest yield of pesticide degrading enzyme was obtained after 48 hours in shake flask culture,and the enzyme activity reached 15.3 U/mL.The optimal reaction temperature of the recombinant enzyme was 35℃,25-45℃ with good thermal stability;The results showed that the optimal pH value of the enzyme was 8.0,pH 7.0-9.0,and the acid-base stability was good;The Michaelis constant K_(m) was 0.076 mmol/L,and the maximum reaction rate V_(max) was 3.023 mmol/(L·min).【Conclusion】It was found that more fenpropathrin degrading enzymes can be produced by Escherichia coli BL21/pET-24 a(K+)-pytZ,with higher purity,wider adaptability to temperature and pH,and higher degradation efficiency.