以万座嗜酸两面菌(Acidianus manzaensis)为研究对象,探索并优化其膜蛋白提取方法,以优化后的方法提取该菌分别以单质硫(S0)和亚铁(Fe2+)为能源底物进行生长时的膜蛋白质,并进行聚丙烯酰胺凝胶电泳(SDS-PAGE)研究膜蛋白在两种能源底物培养下的表达差异。首先通过80℃水浴60-70 min对A.manzaensis胞外黏附蛋白进行初步分离。其次比较了不同提取剂(Triton X-110,SDS和Triton X-114)对膜蛋白的提取效果,结果表明Triton X-114的提取效果较好,其最佳浓度为10%(w/v);比较了不同沉淀剂(三氯乙酸(TCA),丙酮,三氯乙酸/丙酮,甲醇和乙醇)对膜蛋白质沉淀的效果,结果表明TCA/丙酮的沉淀效果最不理想,会导致沉淀后低分子量蛋白发生缺失,而其他几种没有明显差别,综合比较选择较常用的丙酮作为膜蛋白提取的沉淀剂。最后基于优化后膜蛋白提取方法,分别对S0和Fe2+培养的A.manzaensis膜蛋白质进行提取及SDS-PAGE,结果发现分子量为35.6 k D和16.9 k D的蛋白只在A.manzaensis以S0生长时出现,表明这些蛋白质很可能在A.manzaensis硫氧化中发挥了重要作用;分子量为72 k D和26 k D的蛋白质在A.manzaensis以Fe2+生长时比其以S0生长时显著表达上调,表明此蛋白可能与A.manzaensis铁氧化相关。
Iron L-edge and sulfur K-edge X-ray absorption near edge structure（XANES） spectroscopy analysis of pyrite leached by extreme thermophilic Archaea strain Acidianus manzaensis（A.manzaensis） was carried out.Leaching experiments show that the oxidation of pyrite can be accelerated by A.manzaensis.Leaching results show that with the increase of leaching time,pH value in the leaching solution gradually decreases,redox potential increases rapidly from day 0 to day 3,and then increases slowly.The SEM results show that the pyrite surfaces are corroded gradually by A.manzaensis,and the XRD results show that the leaching residues contain new compositions of jarosite and elemental sulfur（S0）.The iron L-edge XANES spectroscopy analysis of pyrite during biooxidation indicates that pyrite is gradually converted to Fe（III）-containing species.The sulfur K-edge XANES spectroscopy analysis indicates that elemental sulfur is produced during bioleaching and maintains mass fractions of 3.2%-5.9%.Sodium thiosulfate was also detected from day 2 to day 4,indicating the existence of thiosulfate during biooxidation of pyrite.
