顺丁烯二酸异构酶工程菌发酵条件优化

Optimization of Fermentation Conditions of Escherichia coli Strain Expressing a Recombinant Maleate cis-trans Isomerase

为提高重组大肠杆菌中顺丁烯二酸异构酶表达量,通过正交试验设计,对工程菌的生长条件和目的蛋白可溶表达条件进行优化.采用250 mL三角瓶中装有50 mL(Amp 100 mg L-1)的培养基,分别研究培养基中葡萄糖、蛋白胨、酵母浸粉的浓度,培养基pH值以及摇床转速、装液量、接种量等对蛋白可溶表达量的影响.确定顺丁烯二酸异构酶工程菌最优化培养基为:蛋白胨20 g L-1、酵母浸粉2.5 g L-1、K2HPO4·3H2O 3.0 g L-1、KH2PO4 1.5 g L-1、NaCl 6 g L-1、MgSO43 g L-1,培养基pH调至6.5.确定顺丁烯二酸异构酶工程菌可溶性表达最优条件为:37℃下培养至D600 nm值为1.0时,添加终浓度为0.05 mmol L-1的IPTG进行诱导,诱导温度37℃,摇床转速220 r min-1,装液量20%,接种量5%,诱导时长为6 h.利用BioFlo 415发酵罐以最优化的培养基和发酵条件对该工程菌进行了3批发酵实验,与摇瓶实验相比,顺丁烯二酸异构酶的表达量提高了近1.5倍,单位发酵液的酶活力由46 U mL-1发酵液提高到78 U mL-1发酵液.以上数据为顺丁烯二酸异构酶重组工程菌的中试发酵奠定了基础.

The fermentation conditions of the Escherichia coli strain [pET32a-Mtil-BL（DE3） 21] expressing a recombinant maleate cis-trans isomerase were optimized by orthogonal experiment design. The effects of the components of the culture medium （tryptone, yeast extract and pH）, shaking speed, medium volume and inoculum concentration on cell growth and expression of maleate cis-trans isomerase were studied in 250 mL erlenmeyer flask containing 50 mL medium. The optimum medium composition was as follows： Tryptone 20 g L^-1, yeast extract 10 g L^-1, K2HPO4-3H2O3.0 g L^-1, KH2PO4 1.5 g L^-1, NaCl 6 g L^-1MgSO4 3 g L^-1, pH 6.5. The optimum conditions for cell growth and expression of maleate cis-trans isomerase were as follows： Inoculum concentration 5%, medium volume 20%, 37 ℃ and shaking speed 220 r min^-1 When D600 reached approximately 1.0, cells were induced by addition of 0.05 mmol L ^-1 oflPTG. Growth was continued at 37 ℃ for up to 6 h. The fermentation was scaled up to 10 L in a BioFlo 415 automatic fermentor. The dissolved oxygen was maintained at about 60%, and the recombinant cells were harvested at 7 h after induction of IPTG. Compared to the shaking-flask experiments, the ratio of target protein to total bacterium proteins increased about 1.5 times, and the enzyme activity increased from 46 U mL^-1 to 78 U mL^-1 These results laid foundation for the scale-up production of recombinant maleate cis-trans isomerase. Fig 7, Tab 2, Ref 17