Purification and characterization of hydrogenase from Synechococcus sp. PCC 7942
XU Hui-juan1, WU Xiao-bing1, LI Xiao-quan1, LONG Min-nan1, LIANG Shi-zhong2
1. School of Life Sciences, Xiamen University, Xiamen 361005, China; 2. College of Biological Science and Engineering, South China University of Technology, Guangzhou 510006, China
Hydrogenase from Synechococcus sp.PCC 7942 was purified to close homogeneity aerobically at room temperature.The hydrogenase-containing crude extract was collected after ultrasonic disruption and removal of cell debris by ultracentrifugation.Subsequently,three steps of column chromatographies(anion exchange,hydrophobic interaction and gel filtration)were performed.Hydrogenase was purified about 218-fold with a yield of 6.5% finally.The purified enzyme has a specific activity for hydrogen evolution of 1.46 U·mg-1 protein.SDS-PAGE gel of the purified enzyme revealed five predominant protein bands with estimated molecular weights of 83,60,47,30 and 27 kDa,respectively.The enzyme is a soluble bidirectional hydrogenase and shows maximum activity while using reduced methyl viologen as an electron donor.The optimum temperature and pH value for hydrogen evolution catalyzed by the purified hydrogenase are 50℃ and pH 8.0.
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