大肠杆菌中1,3-丙二醇氧化还原酶的表达与纯化  

Over-expression of Klebsiella pneumoniae 1,3-Propanediol Oxidoreductase Gene dhaT in Escherichia coli and Purification of the Protein

作　　者：刘海平[1] 郝健[1] 

机构地区：[1]燕山大学环境与化学工程学院,河北秦皇岛066004

出　　处：《生物技术》2011年第1期19-23,共5页Biotechnology

基　　金：国家自然科学基金项目(No.20906076);教育部留学回国人员科研启动基金项目("克雷伯氏肺炎杆菌1;3-丙二醇合成途径还原支路酶促反应动力学研究")资助

摘　　要：目的:在大肠杆菌中表达1,3-丙二醇氧化还原酶(PDOR),并对PDOR进行纯化。方法:从克雷伯氏肺炎杆菌(Kleb-siella pneumoniae)基因组中,克隆PDOR基因dhaT。构建表达载体pDK-dhaT,在E.coli DH5α中利用IPTG诱导进行表达。细胞裂解液利用硫酸铵盐析、Sephadex G-200凝胶层析和DE23 Cellulose阴离子交换层析,进行酶蛋白分离提纯。结果:用SDS-PAGE分析表明胞内PDOR占可溶性蛋白的39.8%,酶活为14.5U/ml。纯化后酶液比酶活提高3.94倍,回收率为15.5%。结论:成功地构建了PDOR高效表达载体,并且得到了高纯度的PDOR。Objective：1,3-propanediol oxidoreductase was expressed in the E.coli DH5α,and was purified.Method：PDOR encoding gene dhaT was cloned from the genome of Klebsiella pneumoniae by PCR.After DNA sequencing,the expression plasmid pDK-dhaT was constructed and transformed into E.coli DH5α.The recombinant strain was cultured and induced with IPTG.PDOR in the lyses of E.coli DH5α（pDK-dhaT） was purified by Ammonium sulfate condensation,Sephadex G-200 gel filtration and DE23 Cellulose ion-exchange chromatography.Result：Analyzed by SDS-PAGE and enzyme activity assay,their show a high level of expression.PDOR accounted for 39.8% of total soluble protein in cytoplasm,and PDOR activity was 14.5 U/ml.The final enzyme obtained has a 3.94-fold purification with the recovery ratio of 15.7%.Conclusion：The PDOR expressing vector was established.PDOR was purified to a high purity.

关 键 词：1 3-丙二醇氧化还原酶 克隆 表达 纯化 

分 类 号：Q554.2[生物学—生物化学]