嗜酸氧化亚铁硫杆菌APS还原酶的表达、纯化及其性质鉴定  

Expression, Purification, and Characterization of APS Reductase from Acidithiobacillus ferrooxidans

作　　者：郑春丽[1,2] 张燕飞[1] 吴安娜[1] 戴云杰[1] 张晓键[1] 柳建设[3,1] 

机构地区：[1]中南大学资源加工与生物工程学院,湖南长沙410083 [2]内蒙古科技大学生物与化学工程学院,内蒙古包头014010 [3]东华大学环境学院,上海201620

出　　处：《现代生物医学进展》2009年第5期812-814,841,共4页Progress in Modern Biomedicine

基　　金：国家973计划"微生物冶金的基础研究"项目(2004CB619201);国家自然科学基金创新研究群体资助项目(50321402);全国优秀博士学位论文作者专项资金资助项目(200549);国家自然科学基金项目(50874032)

摘　　要：嗜酸氧化亚铁硫杆菌(Acidithiobacillus ferrooxidans)中APS还原酶是硫同化途径的一个关键酶,其对硫酸盐的还原及硫化物的氧化具有重要调节作用。本文以A.ferrooxidans ATCC23270基因组为模板,通过PCR扩增得到编码APS还原酶的cysH基因,与原核表达载体pLM1构建重组体,转化入大肠杆菌(Escherichia coli,E.coli)DH5α中,测序正确后,加IPTG诱导表达,用一步亲和层析法纯化出浓度和纯度都较高的APS还原酶。由蛋白颜色和紫外分析,确定其含有一个[Fe4S4]簇作为活性中心。表达产物进行SDS-PAGE分析,证实分子量为28kD。酶活测定表明其具有将APS还原为亚硫酸盐跟AMP的功能。Adenosine 5＇-phosphosulfate （APS） reductase is a key enzyme involved in the pathways of sulfate reduction and sulfide oxidation in Acidithiobacillus ferrooxidans. The gene cysH which encodes APS reductase was identified in whole genome ofA. fer- rooxidans ATCC 23270 and cloned, and then the fragment was linked into the prokaryotic expression vector pLM 1. The recombined ex- pression plasmid was transduced into Escherichia coli DH 5c~. After sequence being identified to be right, the protein was expressed in E. coli in the presence of IPTG, and then the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. The colors of the protein and UV-scanning determined the presence of the [Fe4S4] cluster. The molecular mass of the APS reductase was 28 kD confirmed by SDS-PAGE. Enzyme activity assay indicated that this reductase can catalyze the APS to sulfite and the AMP reaction.

关 键 词：嗜酸氧化亚铁硫杆菌 APS还原酶 cysH 克隆 表达 纯化 

分 类 号：Q939.99[生物学—微生物学]