鲨肝刺激物质类似物在大肠杆菌中的高密度发酵

High cell-density fermentation of shark hepatical stimulator analogue in Escherichia coli

摘要:

为建立重组鲨肝刺激物类似物(r-sHSA)的高密度发酵方法, 本研究在利用单因素实验和均匀设计实验优化摇瓶发酵培养基的组成和浓度以及诱导剂(IPTG)浓度的基础上, 利用5 L发酵罐进行了放大试验, 探讨了补料方式、补料培养基的组成和浓度、诱导剂加入时间和诱导后菌体的收获时间对工程菌生物量和r-sHSA产量的影响。结果表明: 在改良LB培养基(0.97% 甘油, 0.91% 酵母粉, 0.72%胰蛋白胨, 0.782% KH2PO4, 0.267% K2HPO4·3H2O, 0.062% MgSO4·7H2O, 0.5% NaCl, pH 7.0)中, 当pH控制在7.0、溶氧浓度为25%~30%的前提下, 采用指数补料方式加入优化后的补料培养基(620 g/L 甘油, 94.8 g/L胰蛋白胨, 3.3 mL/L 微量元素, 7.5 g/L MgSO4·7H2O)进行培养, 在工程菌的OD600达到23时, 加入终浓度为0.5 mmol/L的IPTG诱导6 h后收获菌体, 菌体的生物量可达(123.27±1.184) g/L, r-sHSA产量为(2.662±0.041) g/L, 比优化前提高了13.7倍。

Abstract:

The potential effects of recombinant shark hepatical stimulator analogue (r-sHSA) in liver disease have been revealed in our previous studies. In order to further evaluate its clinic application, we carried out high cell-density fermentation in 5 L fermentor to get enough products. Based on the trials in shaking flask, we optimized the parameters for 5 L fermentor, including medium composition, medium supplement, inducer concentration and induction time, etc. In detail, the improved LB medium (0.97% glycerol, 0.91% yeast extract, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4·3H2O, 0.062% MgSO4·7H2O, 0.5% NaCl, pH 7.0) is chosen to cultivate the engineering bacteria with the constant fermentation condition (pH 7.0, and the dissolved oxygen concentration is about 25%-30%). When bacterial culture reaches exponential phase, the modified feeding medium (620 g/L glycerol, 94.8 g/L tryptone, 3.3 mL/L trace elements, and 7.5 g/L MgSO4·7H2O) is then supplied through the method of exponential fed-batch mode. After the optical density (OD600) of engineering bacterial culture reaches to 23, the ultimately concentration of 0.5 mmol/L IPTG is added to induce the expression of r-sHSA for 6 h. Results show that the amount of r-sHSA production is (2.662±0.041) g/L, which is about 13.7 folds of the one optimized before.