组成型表达粪产碱杆菌青霉素G酰化酶重组大肠杆菌发酵条件研究

Fermentation Conditions for High-level Constitutive Expression of Alcaligenes faecalis Penicillin G Acylase in Escherichia coli

目的:对重组大肠杆菌组成型表达粪产碱杆菌青霉素G酰化酶(AfPGA)进行了发酵条件研究。方法:在摇瓶和5L发酵罐中研究了(NH4)2SO4和葡萄糖浓度对质粒的分离稳定性及青霉素G酰化酶表达的影响。结果:该工程菌质粒具有分离不稳定性,培养基中无(NH4)2SO4时发酵过程中pH和糊精水解生成葡萄糖的浓度变化较小,细胞前期(0h-12h)的生长速率降低,质粒分离稳定性和青霉素G酰化酶的表达水平提高。发酵过程中维持低葡萄糖水平可以限制细胞的生长速率,提高质粒稳定性和促进青霉素G酰化酶的合成。采用混合碳源发酵,发酵培养基含糊精2g/L,12h后以1g/L.h恒速流加葡萄糖至35h,控制流加过程葡萄糖浓度0.1g/L左右,平均比生长速率为0.06h-1,发酵结束时质粒稳定性为86%,青霉素G酰化酶的表达水平达23 000U/L。结论:重组大肠杆菌组成型表达青霉素G酰化酶的研究对工业生产有一定指导意义。

Objective： Cultivations of recombinant Esherichia coli DH5α constitutively expressing Alcaligenes faecalis penicillin G acylase （AtPGA） were studied.Methods：The influence of ammonium sulfate and glucose on plasmid, stabilimid stability and penicillin G acylase activity were conducted in flask shakes and a 5L fermentor.Results： It was found that the constitutive expression of penicillin G acylase ineased plasmid instability. If the ammonium sulfate was removed from the medium,the fluctuation of pH and glucose concentration （hydrolysate of dextrin）was small in the fermentation. At the same time the cell growth rate was decreased in 0h- 12 h and the plasmid stability and penicillin G acylase activity were increased. In low glucose level, the cell growth rate was lessened, and the plasmid stability and the expression of penicillin G acylase were increased. Mixed carbon sources were used in the fermentation process. The basal medium included 2g/L dextrin and the constant feeding of 1g/L·h glucose started at 12h and stopped at 35h. The glucose concentration was controlled around 0.1g/L and the average specific growth rate was 0.06h^-1. Under the above conditions the plasmid stability reached 86% and penicillin G acylase activity was 23 000U/L. Conclusion： Tne study of constitutive expression of penicllin G acylase in E. coli was a guidance to industrial production.