不同来源胰蛋白酶的基因挖掘、在Komagataella phaffii GS115的异源表达及其酶学性质分析  

Gene mining,heterologous expression and biochemical characterization of trypsin from different sources in Komagataella phaffii GS115

作　　者：何林曼 钮成拓 刘国正 郑飞云[1] 刘春凤[1] 王金晶 李崎[1] HE Linman;NIU Chengtuo;LIU Guozheng;ZHENG Feiyun;LIU Chunfeng;WANG Jinjing;LI Qi(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China)

机构地区：[1]工业生物技术教育部重点实验室(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》2024年第2期100-105,共6页Food and Fermentation Industries

基　　金：国家重点研发计划子课题(2021YFC2100404)。

摘　　要：胰蛋白酶是一种丝氨酸蛋白酶,广泛应用于皮革、食品加工、生化检测和制药行业。该文旨在挖掘不同来源的胰蛋白酶,并在Komagataella phaffii GS115进行异源表达,最终分析并比较其酶学性质。通过基因挖掘手段分别克隆了人源胰蛋白酶(homo spanies trypsin,HST)、牛源胰蛋白酶(bos taurus trypsin,BTT)、虾源胰蛋白酶(penaeus vannamei trypsin,PVT)和弗氏链霉菌源胰蛋白酶(Streptomyces fradiae trypsin,SFT),以pPIC9k为载体构建重组质粒,并在Komagataella phaffii GS115中成功实现异源表达。经纯化激活后,HST,BTT,PVT,SFT的酰胺酶活性分别为18.6、12.3、44.7、13.8 U/mL;4种来源胰蛋白酶的最适温度分别为20、35、40、45℃,而其最适pH值分别为9、8.5、8.5、9,其中SFT在pH 9~11的稳定性更优。Trypsin is a serine trypsin which is widely used in the leather making,food processing,biochemical testing,and pharmaceutical industries.This study aimed to obtain novel trypsin genes from different sources,heterologously expressed in Komagataella phaffii and characterized their enzymatic properties.Through gene mining strategy,potential trypsin genes were obtained from human,bovine,shrimp and Streptomyces fradiae and successfully expressed in the K.phaffii GS115 through pPIC9k vector.After purification and activation,the activities of human-derived trypsin(Homo sapiens trypsin,HST),bovine-derived trypsin(Bos taurus trypsin,BTT),shrimp-derived trypsin(Penaeus vannamei trypsin,PVT)and S.fradiae-derived trypsin(SFT)were 18.6,12.3,44.7,and 13.8 U/mL,respectively.The optimum temperatures of the above four types of trypsin were 20,35,40,and 45℃while their optimum pH values were 9,8.5,8.5,and 9,respectively.Among four types of trypsin,SFT showed the best stability at alkaline environment since it was relatively stable at pH 9-11.

关 键 词：胰蛋白酶 基因挖掘 异源表达 毕赤酵母 酶学性质 

分 类 号：Q789[生物学—分子生物学] TQ925[轻工技术与工程—发酵工程]