重组大肠杆菌分泌表达鼠源羧肽酶B及其培养基优化  

Extracellular expression and fermentation optimization of rat carboxypeptidase B in Escherichia coli

作　　者：於瑞梅 辛瑜[2] 顾正华 吴松[3] 李由然 丁重阳 石贵阳[1,2] 张梁 YU Ruimei;XIN Yu;GU Zhenghua;WU Song;LI Youran;DING Zhongyang;SHI Guiyang;ZHANG Liang(National Engineering Laboratory of Food Fermentation Process and Technology,Jiangnan University,Wuxi 214122,China;School of Biotechnology,Jiangnan University,Wuxi 214122,China;Suzhou Kunpeng Biotechnology Co.,Ltd.,Suzhou 215300,China)

机构地区：[1]江南大学粮食发酵工艺与技术国家工程实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122 [3]苏州鲲鹏生物技术有限公司,江苏苏州215300

出　　处：《生物加工过程》2020年第6期681-689,共9页Chinese Journal of Bioprocess Engineering

基　　金：江苏省重点研发计划(BE2018055)。

摘　　要：为了简化纯化步骤,节约生产成本,笔者构建了能够胞外分泌鼠羧肽酶原B(proCPB)的重组大肠杆菌BL21(DE3)/pET28a-OmpA-proCPB,并进行发酵优化。结果表明:在信号肽OmpA的引导下,proCPB成功分泌至胞外,激活后的羧肽酶B(carboxypeptidase B,CPB)比酶活为131.22 U/mg。通过单因素及正交试验对发酵工艺进行优化,得出最佳培养基组合为甘油7 g/L、复合氮源15 g/L、MgSO_44 mmol/L、山梨醇0.3 mol/L、NaCl 10 g/L;最佳发酵条件为诱导温度30℃,诱导时间24 h。优化后,胞外proCPB的相对表达量为6.35(优化前的相对表达量为1)。首次实现了鼠羧肽酶原B在大肠杆菌中的胞外分泌表达,为羧肽酶B的工业化直接应用提供了一定的技术支持。To simplify the purification process and reduce cost,recombinant strain Escherichia coli BL21(DE3)/pET28 a-OmpA-proCPB was constructed for extracellularly secreting rat procarboxypeptidase B(proCPB)and the fermentation conditions were optimized.Results showed that under the guidance of signal peptide OmpA,proCPB was successfully secreted,and the specific activity of activated carboxypeptidase B(CPB)was 131.22 U/mg.An optimized fermentation strategy was developed through single factor and orthogonal tests.The optimal medium was determined as follows:7 g/L glycerol,15 g/L compound nitrogen,4 mmol/L MgSO4,0.3 mol/L sorbitol and 10 g/L NaCl.The optimal culture conditions were:inducing temperature 30℃and inducing time 24 h.After optimization,the relative expression of extracellular proCPB was 6.35(the relative expression of the initial level was defined as 1).Our findings could provide technical support for the direct industrial application of carboxypeptidase B.

关 键 词：羧肽酶B 分泌表达 大肠杆菌 发酵优化 

分 类 号：Q81[生物学—生物工程]