钝齿棒杆菌乙酰谷氨酸激酶编码基因argB的克隆表达、纯化及酶学性质研究  

Expression, purification and characterization of acetylglutamate kinase for producing arginine from Corynebacterium crenatum

作　　者：刘飞[1] 饶志明[1] 徐美娟[1] 许泓瑜 张晓梅 窦文芳 许正宏 

机构地区：[1]工业生物技术教育部重点实验室,江南大学生物工程学院工业微生物研究中心,江苏无锡214122 [2]江南大学医药学院,江苏无锡214122

出　　处：《工业微生物》2010年第4期23-28,共6页Industrial Microbiology

基　　金：国家"863计划"(No.2007AA022207);国家"973项目"(2007CB707804);国家自然科学基金(No.20676053;30970056);霍英东青年基金(No.121020)

摘　　要：本研究的钝齿棒杆菌(Gorynebacterium crenatum SYPA)是筛选得到的高产精氨酸突变菌株,其中argB基因编码的乙酰谷氨酸激酶(NAGK)是精氨酸合成过程中的关键酶。为了进一步研究该酶的相关特性,以钝齿棒杆菌(Corynebacterium crenatum SYPA)基因组为模板,扩增得到其arB基因,成功构建了pET-28a-argB重组质粒,并在E.coli BL21(DE3)中诱导后高效表达。利用载体pET-28a上的His6·Tag标记选用Ni柱亲和层析法纯化表达具有活性的NAGK,纯化后酶的比活力达到7.28 U/mg,纯化倍数达66.18。并对该酶的酶学性质进行了初步研究,该酶的最适反应温度为30℃;最适pH值为9.0;酶学动力学参数以N-乙酰谷氨酸为底物的Km为3.35mmol/L;金属离子Cu^(2+)、Mn^(2+)、螯合剂对乙酰谷氨酸激酶均有明显的抑制作用。The strain Corynebacterium crenatum SYPA is a mutant strain of high production of arginine, and the NAGK encoded by gene argB is a key enzyme which is feedbackinhibited in its activity by arginine. For studying the characterization of NAGK, the argB gene encoding NAGK in Corynebacterium crenatum SYPA was amplified and in- serted into expression vector pET-28a. Then the plasmid pET-28a-argB was constructed and introduced into Escherichia coli BL21 （DE3）. SDS-PAGE showed that the gene argB was expressed successfully in recombinant E. coli BL21. NAGK was purified by Ni-NTA affinity chromatography. The results showed that a single band was about 34 kD on SDS-PAGE gel, the specific activity was about 7.28 U/mg, and the purification fold was 66.18 times. The optimum activity of NAGK was at pH 9.0 and 30℃. Km of N-acetylglutamate was 3.35 mmol/L. Its activity was obviously inhibited by Cu^2 ＋ , Mn^2 ＋ and EDTA.

关 键 词：钝齿棒杆菌 乙酰谷氨酸激酶 表达 纯化 酶学性质 

分 类 号：Q936[生物学—微生物学]