欧文氏菌SCB125中2-酮醛糖酸还原酶基因在大肠杆菌中的克隆与表达  

Cloning and Expression of 2-Ketoaldonate Reductase Gene in E.coli from Erwinia SCB125

作　　者：陈芳[1] 陈策实[1] 尹光琳[1] 

机构地区：[1]中国科学院上海生物工程研究中心,上海200233

出　　处：《中国医药工业杂志》2001年第5期193-197,共5页Chinese Journal of Pharmaceuticals

基　　金：中国自然科学基金项目! (3 9770 0 2 1)

摘　　要：根据酶活测定和 PAGE活性染色初步证明欧文氏菌 SCB12 5中存在酶活较高的 2 -酮醛糖酸还原酶 (2 - KR) ,能够以 2 ,5 -二酮 - D-葡萄糖酸 (1)、2 -酮 - L -古洛糖酸 (2 )或者 2 -酮 - D-葡萄糖酸 (3)为底物。抽提欧文氏菌染色体DNA为模板 ,利用 PCR技术扩增 ,克隆得到两个 2 - KR酶基因 (tkr A和 tkr B) ,通过酶切和测序证明 :2 - KR A基因发生多处突变 ,而 2 - KR B基因保守。将含有这两个基因的片段分别连接到表达载体 p BL并转化大肠杆菌 DH 5α后 ,均获得高酶活表达。如果进一步用基因剔除的方法破坏欧文氏菌染色体上野生型 2 - KR基因 ,可以获得一个表达 1还原酶的理想宿主 ,为实现从葡萄糖一步发酵生产维生素 C前体There are two 2 ketoaldonate reductases with high activity in Erwinia SCB125. It was identified by enzyme activity analysis and native gel activity stain. The chromosome DNA of Erwinia SCB125 was extracted and used as template, and two genes( tkrA and tkrB )encoding these two reductases were amplified and cloned into pGEM T vector. Sequence analysis showed that there were mutants for 2 KR A gene only. They were cloned into expression vector pBL and were successfully expressed with high enzyme activity in E.coli DH5α. It was tested by gene knocking out technique to block these two 2 ketoaldonate reductases genes.

关 键 词：2-酮醛糖酸还原酶 欧文氏菌 PCR 克隆 基因表达 基因剔除 维生素C 前体药 

分 类 号：TQ466.3[化学工程—制药化工] Q78[生物学—分子生物学]