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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5L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,pH7.0)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(620g/L甘油,94.8g/L胰蛋白胨,3.3mL/L微量元素,7.5g/LMgSO4·7H2O)进行培养,在工程菌的OD600达到23时,加入终浓度为0.5mmol/L的IPTG诱导6h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。
In order to establish a high-density fermentation method for r-sHSA, a single factor experiment and a uniform design experiment were used to optimize the composition and concentration of shake flask fermentation medium and the concentration of IPTG On the use of 5L fermenter amplification test carried out to explore the feeding mode, the composition and concentration of feed medium, inducer time and the induction of cell harvest time on the impact of engineering bacteria biomass and r-sHSA yield . The results showed that in the modified LB medium (0.97% glycerol, 0.91% yeast powder, 0.72% tryptone, 0.782% KH2PO4, 0.267% K2HPO4.3H2O, 0.062% MgSO4.7H2O, 0.5% NaCl, pH 7.0) Under the premise of pH control of 7.0 and dissolved oxygen concentration of 25% ~ 30%, the optimized feed medium (620 g / L glycerol, 94.8 g / L tryptone, 3.3 mL / L trace element , 7.5g / L MgSO4 · 7H2O). After the OD600 of the engineered bacteria reached 23, the cells were harvested by IPTG with a final concentration of 0.5mmol / L for 6h. The biomass of the cells reached 123.27 ± 1.184g / L and the yield of r-sHSA was (2.662 ± 0.041) g / L, which was 13.7 times higher than that before optimization.