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

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.