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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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Citing This Article：

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).