海鲍分离微生物Acinetobacter sp.酯酶基因的表达和酶学性质  

Expression and characterization of an alkaline esterase from marine Acinetobacter sp.

作　　者：张秋芳[1] 李仁宽[1,2] 林娟[1,2] 叶秀云[1,2] 

机构地区：[1]福州大学生物科学与工程学院,福建福州350108 [2]酶高效表达国家工程实验室,福建福州350002

出　　处：《微生物学通报》2014年第9期1785-1792,共8页Microbiology China

基　　金：海洋公益性行业科研专项项目(No.201305015);国家863计划项目(No.2013AA102101);福建省教育厅项目(No.JA13032);福建省自然科学基金项目(No.2011J01218)

摘　　要：【目的】克隆源于海鲍内脏中一株不动杆菌Acinetobacter sp.的酯酶基因estA,并对其进行重组表达和性质研究。【方法】利用分子生物学技术克隆出酯酶基因estA并构建pPICZα-C-estA重组表达载体,并通过电转化方法将重组质粒转入毕赤酵母X33中;通过甲醇诱导培养重组菌获得重组酯酶,并对重组酯酶进行生化表征。【结果】克隆得到的estA基因序列全长912 bp,编码304个氨基酸;重组X33发酵上清液中酯酶酶活力达到1 200 U/L,重组酯酶的分子量约为33.7 kD;酶学性质研究表明重组酯酶催化底物对硝基苯乙酸乙酯水解反应的最适pH和温度为8.0和40?C,在pH 8.0-10.0温度及小于60?C时具有较好的稳定性。【结论】成功克隆了海洋来源的不动杆菌酯酶基因并在Pichia pastoris中实现了高效表达。[Objective] The gene encoding estA in a strain of Acinetobacter sp. screened from abalone visceral was cloned and expressed in Pichia pastoris. [Methods] The estA gene was amplified and integrated into the genome of P. pastoris X33 via the pPICZα-C vector. The recombinant estA was expressed into the culture medium, and the recombinant protein were purified and characterized.[Results] The estA gene of 912 bp was cloned. After induced with methanol, recombinant esterase reached 1 200 U/L with weight of 33.7 kD. The optimum pH and temperature of recombinant esterase were 8.0 and 40 ？C, respectively. Furthermore, this esterase exhibited remarkable stability at pH between 8.0-10.0 and under 60 ？C. [Conclusion] The gene of estA from Acinetobacter sp. was successfully cloned and expressed in P. pastoris.

关 键 词：不动杆菌 酯酶 毕赤酵母 克隆表达 

分 类 号：Q78[生物学—分子生物学]