海洋细菌有机磷酸酐水解酶在大肠杆菌中的分泌表达研究  

Expression of a marine bacterial organophosphorus acid anhydrolase in Escherichia coli

作　　者：肖运柱[1,2] 杨键[1,2] 张偲[1,2] 龙丽娟[1,2] 

机构地区：[1]中国科学院南海海洋研究所热带海洋生物资源与生态重点实验室RNAM海洋微生物中心广东省海洋药物重点实验室,广东广州510301 [2]中国科学院大学,北京100049

出　　处：《生物加工过程》2017年第3期1-6,共6页Chinese Journal of Bioprocess Engineering

基　　金：国家自然科学基金青年基金(41406193);广东海洋经济创新发展区域示范项目(GD2012-D01-002);中科院战略性先导科技专项(XDA10030404;XDA13020301)

摘　　要：为了考察一种海洋细菌来源的有机磷酸酐水解酶OPAA4301在大肠杆菌中胞外分泌表达生产的可能性,笔者分别从信号肽和发酵条件两方面对该酶在大肠杆菌中的产量及细胞定位情况进行观测和优化。结果发现,海洋细菌有机磷酸酐水解酶OPAA4301在大肠杆菌中可不依赖信号肽少量分泌至培养基上清中。以胞外酶产量为指标,对诱导剂和甘氨酸添加量进行优化,确定适宜的诱导剂浓度为0.8 mmol/L,适宜甘氨酸添加量为10 g/L。在优化条件下,重组酶蛋白的细胞定位主要由细胞内转变为细胞外,胞外酶活提高108.8倍,达0.28 U/m L,相当于重组蛋白胞外生产能力为0.72 g/L。本研究为重组海洋细菌有机磷酸酐酶的工程化发酵生产提供理论基础。To realize the expression of recombinant marine bacterial organophosphorus acid anhydrolase 0 PAA 4301 in Escherichia coli,we optimized fermentation production. Two factors,signal peptide and culture condition,were assumed critical in secretion of recombinant enzymes. 0 ur results showed that detectable organophosphorus acid hydrolase was found in the culture supernatant of recombinant E . coli constructed without signal peptide at the N-terminal of the enzyme.Using the secretory expression level as target,we studied the effects of supplementation of inducer and glycine.The optimized concentrations of inducer and glycine were determined to be 0. 8 m m o l / L and 10 g/L.Under the optimized conditions,the cellular localization of recombinant enzyme was converted from intracellular to extracellular,and the extracellular enzyme expression level improved by 108. 8 folds.The maximum extracellular enzyme activity reached 0.28 U /mL,which is the equivalent of 0.72 g/L for the production capacity of recombinant enzyme. 0 ur study can provide theoretical basis for producing recombinant marine bacterial organophosphorus acid anhydrolase.

关 键 词：海洋细菌 有机磷酸酐水解酶 蛋白质分泌表达 大肠杆菌 甘氨酸 

分 类 号：Q851[生物学]