花生根瘤菌类菌体氢酶的分离提纯及特性

Purification and Some Properties of the Membrane-Bound Hydrogenase from the Bacteroids of Peanut Root Nodules

花生根瘤菌类菌体经超声波破碎，ＴｒｉｔｏｎＸ－１００溶解，正已烷－硫酸铵处理后，再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤，获得凝胶电泳纯的膜结合态氢酶，比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１，为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后，呈现两个蛋白带，分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体，其中以ＤＣＩＰ为最适。

The H2-uptake hydrogenase fromthe bacteroids of peanut nodules hasbeen purified and characterized. Bacteroids were prepared, then broken bysonication. The membrane-bound hydrogenase was solubilized by treatmentwith Triton X-100 and followed byammonium sulfate-he-cane extractionto remove lipids and detergent.. Thehydrogenase was further purified byDEAE-cellulars column chromatography, eluted with Tris-HCl buffer containing NaCl 0. 3 mol/L and GenapolX-080 0. 2%. The enzyme was purified to homogeneity by runningSephacryl H200 column chromatogtaphy twice. The specific activity was71. 4 pmol H2 oxidiZed Per mg proteinper mid and was incrsased 211 foldsrelative to that in hacteroids. Themolecular weisht of native enzyme wasabout 1 10 kD as determined by PAGEin undenatured form. SDS-PAGEShowed two bands with molecularweight of 65 kD and 35 kD, indicatingthat the membrane-bound hydsogenasewas an a, 6 dimer. The nickel contentof purified hydrogenase was found tobe 0. 62 mole Ni/mol hydrogenase.The optimum PH for the activity of thepurified enzyme was found to be near6. 5 in potassium phosphate buffer.Suitable electron acceptors are DCIP,methylene blue, ferricyanide and CytC. The best one is DCIP. Benzyl viologen, methyl viologen and NAD werealmost not reduced.