Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中分泌途径的鉴定及其分泌能力的提高  

Secretion Pathway Identification of Pyrococcus yayanosii L-Asparaginase in Bacillus subtilis and the Secretion Level Improvement

作　　者：李谞 徐书琴 张显[1,2] 徐美娟 杨套伟[1,2] 张惠玲 方海田[3] 饶志明 LI Xu;XU Shuqin;ZHANG Xian;XU Meijuan;YANG Taowei;ZHANG Huiling;FANG Haitian;RAO Zhiming(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;School of Agriculture,Ningxia University,Yinchuan 750021,China)

机构地区：[1]江南大学工业生物技术教育部重点实验室,江苏无锡214122 [2]生物工程学院,江苏无锡214122 [3]宁夏大学农学院,宁夏银川750021

出　　处：《食品与生物技术学报》2020年第11期34-40,共7页Journal of Food Science and Biotechnology

基　　金：国家自然科学基金项目(31500065);国家轻工技术与工程一流学科自主课题项目(LITE2018-06);中央高校基本科研业务费专项资金项目(JUSRP51708A);宁夏回族自治区科技创新团队项目(KJT2017001);宁夏回族自治区重点研发计划项目(2017BY069);江苏高校品牌专业建设工程资助项目;江苏高校优势学科建设工程资助项目。

摘　　要：食品安全(GRAS)菌株枯草芽孢杆菌由于其具有良好的分泌表达能力及易于基因工程操作等特性被广泛用作外源蛋白质的表达菌株。枯草芽孢杆菌中蛋白质的分泌大多依赖于信号肽介导的Sec分泌途径(General secretion pathway)和Tat分泌途径(Twin-arginine translocation pathway)。在本研究中,通过信号肽预测、蛋白质N端测序等手段发现来自于Pyrococcus yayanosii L-天冬酰胺酶在枯草芽孢杆菌中的分泌并不依赖信号肽,该酶通过非经典蛋白质分泌途径(non-classical protein secretion pathway)进行分泌。通过信号肽筛选,发现最适合该酶在枯草芽孢杆菌中表达的信号肽为Tat分泌途径信号肽SPphoD,并通过共表达分子伴侣PrsA的方式将该L-天冬酰胺酶的分泌量提高了72.11%。Bacillus subtilis,generally recognized as safe(GRAS),waswidely used as host strain to express heterologous proteins due to its good secretion ability and easy geneticmanipulations.In B.subtilis,most of the signal peptide-mediated secretion was depended on Sec-pathway(General secretion pathway)or Tat-pathway(Twin-arginine translocation pathway).In this study,through signal peptide prediction and protein N terminal sequencing,the secretion of Pyrococcus yayanosii L-asparaginase in B.subtilis was confirmedto depend onnon-classical protein secretion pathway.By signal peptidescreening,the Tat-pathway signal peptide SPphoD was found to bebeneficial for the L-asparaginase secretion in B.subtilis.With the co-expression of SPphoD and molecular chaperone PrsA,the L-asparaginase secretion was increased by 72.11%compared with that of the original one.

关 键 词：枯草芽孢杆菌 分泌表达 L-天冬酰胺酶 信号肽 分子伴侣 

分 类 号：Q933[生物学—微生物学]