| 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 |
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| 引用本文： | 陈芳,陈策实,尹光琳. 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[J]. 中国医药工业杂志, 2001, 32(5): 193-197 |
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| 作者姓名： | 陈芳 陈策实 尹光琳 |
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| 作者单位： | 中国科学院上海生物工程研究中心, |
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| 基金项目： | 中国自然科学基金项目(39770021) |
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| 摘 要： | 根据酶活测定PAGE活性染色初步证明欧文氏菌SCB125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）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(tkrA和tkrB)通过酶切和测序证明：2－KR A基因发生多处突变，而－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PBL并转化大肠杆菌DH5α后，均获得高酶活表达，如果进一步用基因锡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2打下基础。
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| 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 |
| 文章编号： | 1001-8255(2001)05-0193-05 |
| 修稿时间： | 1999-12-02 |
Cloning and Expression of 2-Ketoaldonate Reductase Gene in E.coli from Erwinia SCB125 |
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| CHEN Fang,CHEN Ce-Shi,YIN Guang-Lin. Cloning and Expression of 2-Ketoaldonate Reductase Gene in E.coli from Erwinia SCB125[J]. , 2001, 32(5): 193-197 |
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| Authors: | CHEN Fang CHEN Ce-Shi YIN Guang-Lin |
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| Abstract: | 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. |
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| Keywords: | 2-ketoaldonate reductase cloning expression knock-out 2-KLG |
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