有机磷水解酶的大肠杆菌细胞表面展示与酶活特性

有机磷水解酶的大肠杆菌细胞表面展示与酶活特性 Cell-Surface Display of Organophosphorus Hydrolase in Escherichia coli and Its Whole-Cell Catalytic Activity 张红星 ZHANG Hongxing 1 2 first-author 李茜茜 LI Qianqian 1 2 叶婷 YE Ting 1 2 郑世学 Zheng Shixue 1 2 李林 LI Lin 1 2 华中农业大学农业微生物学国家重点实验室 华中农业大学农业微生物学国家重点实验室 State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University State Key Laboratory of Agricultural Microbiology,Huazhong Agricultural University 华中农业大学农业微生物学国家重点实验室,湖北,武汉,430070 华中农业大学农业微生物学国家重点实验室,湖北,武汉,430070 利用丁香假单胞菌（Pseudomonas syringae）KCTC1832菌株的冰核蛋白（InaK）的N-末端作为锚定单元和来自黄杆菌（Flavobacterium sp.）ATCC27551菌株opd基因编码的有机磷水解酶作为功能蛋白,构建了一株具有全细胞催化效应的大肠杆菌（Escherichia coli）表面展示工程菌MMBL-405.对该工程菌全细胞有机磷水解酶活性的正交试验结果表明,在诱导物IPTG浓度为0.1 mmol/L、诱导温度为20℃、诱导时间为8 h和Co2+添加浓度为100μmol/L的优化培养条件下,其全细胞酶活性可达到0.62 U/mg细胞干重,是目前国外同类工作所报道酶活性的13.7倍以上.对工程菌所携带的外源质粒在无抗性条件下的稳定性进行了测定,结果表明工程菌经连续转接7次和继代培养168 h后,质粒携载率仍达到60%. In the present study,the N-terminal domain of ice nucleation protein InaK from Pseudomonas syringae KCTC1832 strain was used as an anchoring motif,to display organophosphorus hydrolase（OPH） that was encoded by opd gene of Flavobacterim sp.ATCC27551 strain,on the cell surface of Escherichia coli JM109 strain.Under an optimized culture condition by induction with 0.1 mmol/L of IPTG for 8 h at 20 ℃,and supplementing 100 μmol/L of Co2+,it was found that the engineering strain,MMBL-405,exhibited a maximum whole-cell activity of OPH,which reached up to 0.62 U per mg of cell dry weight,and was at least 13.7-fold higher in whole-cell OPH activity compared to the current similar attempts in the literatures.Additionally,by determining the successive growth of MMBL-405 in the absence of the antibiotic,it showed that the stability of the introduced plasmid was 60% when inoculating in succession for 7 times and culturing for 168 h.It is promising therefore to further develop a live system for biodegradation of organophosphorus compound based on such a strategy. 有机磷水解酶 表面展示 生物降解 全细胞酶活性 organophosphorus hydrolase cell surface display biodegradation whole cell activity Q789 国家自然科学基金资助项目（30370026） 2008-02-01 2021-04-01 2