产菊酯降解酶重组菌的高密度发酵工艺优化  

Enhancing production of a novel pyrethroid-hydrolyzing enzyme by high density fermentation of Escherichia coli

作　　者：范新炯[1,2] 莫忠兴 张曼曼 裴生斌 刘玉焕[2] 刘孝龙[1,2] FAN Xinjiong;MO Zhongxin;ZHANG Manman;PEI shengbin;LIU Yuhuan;LIU Xiaolong(School of Basic Medical Sciences,A n h u i Medical U n iv e rs i ty,Hefei 2302;School of Life Sciences,Sun Yat-sen University,Guangzhou 510275,C h in a)

机构地区：[1]安徽医科大学基础医学院,安徽合肥230032 [2]中山大学生命科学学院,广东广州510275

出　　处：《中山大学学报（自然科学版）》2018年第3期108-119,共12页Acta Scientiarum Naturalium Universitatis Sunyatseni

基　　金：国家自然科学基金(31400680);安徽医科大学校科研基金(2017Xkj007)

摘　　要：为提高菊酯降解酶的产量,利用单因素和正交实验法对重组菌Escherichia coli BL21(DE3)/p ET-28a(+)-sys410的发酵培养基组成和发酵条件进行优化。结果显示最优培养基含有20.0 g·L-1甘油、20.0 g·L-1酵母粉、8.0 g·L-1硫酸铵、100.0 mmol·L-1磷酸盐、5.0 mmol·L-1硫酸镁,以及1.5 m L·L-1的微量元素;发酵条件为装液量50 m L/250 m L、初始培养基pH 7.0、接种量2%,接种培养8 h后乳糖诱导6 h。在7.5 L发酵罐上,使用低浓度碳氮源进行培养,前期恒速补料,对数中后期进行乳糖诱导,后期恒溶氧补料,最终菌体密度和酶活力分别为142.6和3 105.1 U·m L-1,较摇瓶发酵分别提高13.7倍和31.9倍。发酵液经破碎和冷冻干燥后酶活力达到119 426.9 U·g-1,且该酶对菊酯的降解率高于95%。We previously screened and obtained a novel pyrethroid-hydrolyzing gene 410from Turban Basin metagenomic library , which was expressd in E. coli BL21 （ DE3 ） in soluble form. Her , to enhance the production and activity of the enzyme , flask-shaking medium and fermentation condition were investigated. The optimum medium was as follows ： glycerol 20. 0 g·L-1 , yeast extract 20. ammonium sulfate 8. 0 g · L-1 , phosphate 100. 0 mmol · L-1 , magnesium sulfate 5. 0 mmol·L-1 , trace element solution 1. 5 mL·L-1. The optimum conditions of flask-shaking fermentation were as follows ： loading volume 50/250 （V/V） , initial medium pH 7. 0 , inoculum size 2% , lactose induction We also studied high-density fermentation of the recombinant strain in 7. 5 L fermenter. The results showed that dispersed oxygen supply , low concentration of the initial medium , and carbon fed-batch culture could stimulate the growth of the recombinant strain. Inducible expression middle and later stages of logarithmic growth with 6 g·L-1 lactose at 37 0C. After 10 h , cell density reached 142. 6 and the enzymatic activity was 3 105. 1U·mL-1 , 13. 7 and 31.9 times better than those of the flask-shaking fermentation , respectively. In addition , crude enzyme powder was obtained after sonication and freeze dryig with the amount of 68. 4 g·L-1 of fermentation broth. The specific enzyme activity was 119 426. 9U· g-1. Remarkably, the enzyme was able to efficiently degrade all tested pyrethroids within 15 min, reaching over 95% conversion.

关 键 词：农药降解酶 高密度发酵 拟除虫菊酯 生物降解 

分 类 号：Q815[生物学—生物工程]