不同来源的谷氨酸棒杆菌氨基脱氧分支酸合成酶的活性分析  

The Activity Study of Aminodeoxychorismate Synthase of Differernt Corynebacterium glutamicum

作　　者：任建洪[1,2] 张晓梅 窦文芳 许泓瑜 许正宏[1,2] 

机构地区：[1]江南大学医药学院制药工程研究室,无锡214122 [2]江南大学教育部工业生物技术重点实验室,无锡214122

出　　处：《中国生物工程杂志》2009年第8期57-61,共5页China Biotechnology

基　　金：国家"863"计划(2007AA02Z207);国家"973"重点项目(2007CB707804);教育部新世纪优秀人才支持计划(NCET-07-0380)资助项目

摘　　要：分别以高产L-丝氨酸的谷氨酸棒杆菌(Corynebacterium glutamicum)SYPS-062与模式菌株谷氨酸棒杆菌(Corynebacterium glutamicum)ATCC13032的基因组DNA为模板,运用PCR技术扩增出氨基脱氧分支酸合成酶(ADC synthase)的编码基因pabAB。实验结果表明:来源于SYPS-062和ATCC13032的pabAB片段全长均为1863bp,编码620个氨基酸。两片段存在16个碱基的差异,引起了7个氨基酸的突变。将pabAB连接表达载体pET-28a(+),构建表达质粒pET-28a-pabAB,并转化E.coliBL21(DE3),在IPTG诱导下,E.coliBL21(DE3)(pET-28a-pabAB)高效表达分子量约为67kDa的可溶性蛋白。表达产物带有His-tag标记,选用Ni柱对表达产物进行纯化,纯化后酶活测定结果表明,来源于SYPS-062氨基脱氧分支酸合成酶的比酶活低于ATCC13032达46.6%。The two pabAB genes encoding aminodeoxychorismate synthase（ADC synthase） from a L-serine producing strain Corynebacterium glutamicum SYPS-062 and model strain Corynebacterium glutamicum ATCC 13032 were ampilified by PCR. The result of nucleotide sequence analysis showed that both pabAB fragments were 1863bp, encoding 620 amino acids. 16 bases differences that resulted in the changes of 7 amino acids were found in the pabAB of SYPS-062. The two pabAB were inserted into pET-28a to yield the recombinant expression vector pET-28a-pabAB and then transfromed into BL21 （DE3）. Upon IPTG induction, soluble ADC synthase was over-produced by E. coli BL21 （ DE3 ） harboring the expression construct. Recombinant ADC synthase purified by Ni-NTA affinity chromatography showed a single band about 67kDa on SDS-PAGE gel, and activity of aminodeoxychorismate synthase analysis show that the enzyme specific activity of SYPS-062 is 46.6% lower than ATCC 13032.

关 键 词：L-丝氨酸 谷氨酸棒杆菌 pabAB 氨基脱氧分支酸合成酶 

分 类 号：Q819[生物学—生物工程]