毕赤酵母菌分泌表达重组抑肽酶的高密度发酵工艺研究

Study on high-density fermentation technology of expressing aprotinin from Pichia pastoris

目的:对毕赤酵母高密度表达重组抑肽酶的发酵工艺进行探讨。方法:先通过摇瓶培养方法,确定表达抑肽酶的毕赤酵母在BSM培养基中生长所需的最佳配方。在此基础上,采用分批补料培养方法,对毕赤酵母pSA/GS115进行高密度发酵。紫外吸收法测定重组抑肽酶竞争性抑制胰蛋白酶对底物BAEE的水解活性。结果:BSM培养基中毕赤酵母生长必需的最佳配方为甘油40 g.L-1、氨水单次补加量0.1%、甲醇流加量1%。经过毕赤酵母高密度发酵,发酵液上清中蛋白的表达量为37.08 mg.L-1,目的蛋白活性达到4 450 BAEEU.ml-1,比摇瓶培养表达提高了8倍。结论:本发酵工艺可实现酵母工程菌的高密度表达发酵,其发酵产物具有较高的生物活性,为下一步大规模化制备抑肽酶奠定了基础。

Objective： To evaluate the fermentation process of high-density expressed recombinant aprotinin by Pichia pastoris.Methods： The most optimal formulation of BSM medium for Pichia pastoris to grow using flask culture methods were determined.Based on the study of fermentation in shake flask,the optimal conditions were used for scaling up in 7 L fermentor.Engineering bacteria pSA/GS115 was fermented in fed-batch high density fermentation.The biological activity was determined by ultraviolet absorption assay,and the recombinant aprotinin competitively inhibited the hydrolytic activity of trypsin against substrate BAEE.Results： The best technological parameters of fermentation to BSM medium were glycerol 40 g·L-1,ammonia water 0.1%,methanol 1%.Afterbeing optimized,the yield of protein could reach up to 37.08 mg·ml-1.The biological activity determined was 4 450 BAEEU·ml-1,8 folds higher compared to that of shaking flask.Conclusion： The engineering bacteria is successfully fermented in fed-batch high-density fermentation and fermentation produce has a high biological activity,which established a foundation for the large-scale production of aprotinin.