采用组合策略提高灰色链霉菌胰蛋白酶在毕赤酵母中的表达  

High-level production of Streptomyces griseus trypsin by recombinant Pichia pastoris using a combined strategy

作　　者：彭文坚 张娟[2] 刘松 PENG Wenjian;ZHANG Juan;LIU Song(National Engineering Laboratory for Cereal Fermentation Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]粮食发酵工艺与技术国家工程实验室(江南大学),江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《食品与发酵工业》2021年第20期15-21,共7页Food and Fermentation Industries

基　　金：国家重点研发计划项目(2017YFB0308401);国家自然基金面上项目(32071474)。

摘　　要：胰蛋白酶是一种重要的工业酶,广泛应用于皮革加工、食品加工和医药等领域。该研究从菌株改造及发酵条件优化两方面提高了灰色链霉菌胰蛋白酶(Streptomyces griseus trypsin,SGT)在毕赤酵母(Pichia pastoris)中的发酵产量。首先,分析了4种分子伴侣(Cne1、Bmh2、Ssa4和Vhb)和2种转录因子(Yap1、Aft1)对SGT在P.pastoris GS115中表达的影响。摇瓶发酵结果显示,与转录因子Aft1共表达时SGT酶活力较对照菌提高了19.71%,达到217.30 U/mL;其他转录因子或分子伴侣的共表达降低了SGT产量。其次,确定了菌株PP-SGTm/Aft1的最适生长pH和最适诱导pH分别为5.5和6.5。最后,优化了PP-SGTm/Aft1在5 L罐中诱导产酶过程中流加混合碳源(甲醇-山梨醇)的比例。结果显示,当补加的m(甲醇)∶m(山梨醇)=20∶0.5时,5 L罐SGT酶活力达到了2667.24 U/mL(144 h),较仅流加甲醇酶活力提高了20.1%。研究结果将为SGT工业化发酵生产提供重要基础数据。Trypsin is an important industrial enzyme,which is widely used in leather bating,food processing and medicine.The production of Streptomyces griseus trypsin(SGT)by recombinant Pichia pastoris was enhanced by strain modification and optimization of fermentation conditions.Firstly,the effects of four chaperones(Cne1,Bmh2,Ssa4 and Vhb)and two transcription factors(Yap1 and Aft1)on the expression of SGT in P.pastoris GS115 were analyzed.The results of shake flask fermentation showed that when co-expressing with transcription factor Aft1,the SGT activity was 19.71%higher than that of the control strain,reaching 217.30 U/mL.The co-expression of other transcription factors or chaperones reduced SGT production.Secondly,the optimal growth pH and induction pH of PP-SGTm/Aft1 were 5.5 and 6.5,respectively.Finally,the ratio of methanol-sorbitol in 5 L fermentation was optimized.When the ratio of methanol to sorbitol was 20∶0.5,the activity of SGT reached 2667.24 U/mL(144 h),which was 20.1%higher than that of adding methanol only.The results will provide important basic data for industrial fermentation of SGT.

关 键 词：胰蛋白酶 毕赤酵母 分子伴侣 转录因子 发酵优化 混合碳源 

分 类 号：TQ925[轻工技术与工程—发酵工程]