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花生根瘤菌类菌体氢酶的分离提纯及特性

黄南仲, 许良树, 张凤章, 龙敏南

厦门大学生物学系. 福建,厦门 361005

摘要

花生根瘤菌类菌体经超声波破碎, Triton X-100溶解,正巳烷-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71.4#mu#mol H_(2)mg~(-1)prot min~(-1), 为类菌体吸H_(2)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, 分子量分别为65kD和35kD。纯酶的Ni含量为0.62mol Ni/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, 以DCIP最适。图2表3参12

关键词： 花生; 类菌体; 氨酶; 提纯; 特性

Purification and some properties of the membrane-bound hydrogenase from the bacteroids of peanut root nodules

Huang Nanzhong, Xu Liangshu, Zhang Fengzhang, Long Minnan

Department of Biology, Xiamen University. Xiamen 361005,Fujian

Abstract

The H_(2)--uptake hydrogenase from the bacteroids of peanut nodules has been purified and characterized. Bacteroids were prepared, then broken by sonication. The membrane--bound hydrogenase was solubilized by treatment with Triton X--100 and followed by ammonium sulfate--hexane extractionm to remove lipids and detergent. The hydrogenase was further purified by DEAE--cellulose column chromatography, eluted with Tris--HCl buffer containing NaCl 0.3mol/L and Genapol X--080 0.2%. the enzyme was purified to homogeneity by running Sephacryl S--200 column chromatography twice. The specific activity was 71.4 #mu#mol H_(2) oxidized per mg protein per min and was increased 211 folds relative to that in bacteroids. The molecular weight of native enzyme was about 110kD as determined by PAGE in undenatured form. SDS--PAGE showed two bands with molecular weight of 65kD and 35kD,indicating that the membrane--bound hydrogenase was an #alpha#,#beta# dimer. The nickel content of purified hydrogenase was found to be 0.62mole Ni/mol hydrogenase. The optimum pH for the activity of the purified enzyme was found to be near 6.5 in potassium phosphate buffer.

Key words: Penut;Bacteroid;Hydrogenase;Purification;Properties

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引用本文：

黄南仲, 许良树, 张凤章, 龙敏南. 花生根瘤菌类菌体氢酶的分离提纯及特性[J]. 生理学报 1995; 47 (1): .

Huang Nanzhong, Xu Liangshu, Zhang Fengzhang, Long Minnan. Purification and some properties of the membrane-bound hydrogenase from the bacteroids of peanut root nodules. Acta Physiol Sin 1995; 47 (1): (in Chinese with English abstract).