蛋白酶枯草芽孢杆菌工程菌株WB800N/pHT43-npr-PrsA的发酵工艺优化

Optimization of fermentation process for Bacillus subtilisWB800N/pHT43-npr-PrsA recombinant strain with protease activity

试验旨在优化枯草芽孢杆菌工程菌株WB800N/pHT43-npr-PrsA的发酵蛋白酶活性,为国内饲用蛋白酶企业提供技术支持。试验对发酵过程进行葡萄糖含量和比消耗速率监测,确定碳源、氮源对该菌株葡萄糖消耗的动态影响。通过单因素和响应面试验对发酵培养基成分进行优化。结果显示:以0~10 h菌体OD_(600nm) 值为参数优化发酵培养基,以促进菌体生长,获得更高菌体量。10 h以后以酵母粉5 g/L、蛋白胨70 g/L进行补料,延长稳定期,以达到最大单位酶活性。优化发酵培养基的培养条件为:酵母粉60 g/L、蛋白胨30 g/L、消旋葡萄糖10 g/L、MgSO_(4) 3 g/L,发酵8 h时,添加0.5‰异丙基-β-D-硫代半乳糖苷(IPTG)和2‰蛋白酶抑制剂,最终发酵蛋白酶活性为1156 U/mL。研究表明,延迟稳定期能够提高培养基中蛋白胨含量,有利于提高菌体对葡萄糖比消耗速率,蛋白胨对菌体适应周围环境有促进作用,发酵过程中维持葡萄糖浓度处于较低的水平,可以有效避免乙酸生成,促进菌体生长和提高产酶效能,使菌体对底物的消耗保持最旺盛状态。

The experiment aimed to optimize the fermentation protease activity of the engineered Bacillussubtilis strain WB800N/pHT43-npr-PrsA,providing technical support for domestic feed protease enterprises.The fermentation process was monitored for glucose content and specific consumption rate to determine the dynamic impact of carbon and nitrogen sources on the glucose consumption of this strain.The components of the fermentation medium were optimized through single-factor and response surface experiments.The results showed that optimizing the fermentation medium with the OD_(600nm) value of the cell body from 0 to 10 hours as a parameter can promote cell growth and achieve a higher cell quantity.After 10 hours,supplementing with 5 g/L yeast extract and 70 g/L peptone could extend the stationary phase to achieve the maximum specific enzyme activity.The optimized fermentation medium conditions were:60 g/L yeast extract,30 g/L peptone,10 g/L racemic glucose,and 3 g/L MgSO_(4) .At 8 h of fermentation,adding 0.5‰IPTG and 2‰protease inhibitor,the final fermentation protease activity reached 1156 U/mL.The study indicates that delayed stable period can increase the peptone content in the medium,which is beneficial for improving the specific glucose consumption rate of the cells.Peptone promotes the adaptation of the cells to the surrounding environment.Maintaining a low glucose concentration during the fermentation process can effectively avoid the formation of acetic acid,promote cell growth,and enhance enzyme production efficiency,keeping the cell consumption of the substrate at its most vigorous state.