采用硫酸铵沉淀和柱层析(DEAE-Sepharose F.F.,Sephacryl S-200,TSK-DEAE)初步分离纯化了产酸克雷伯氏菌(Klebsiella oxytoca)HP1可溶性氢酶,研究了温度、pH值、电子载体等对氢酶催化放氢活性的影响.研究表明,可溶性氢酶催化放氢的最适温度为30℃,催化放氢的最适pH值为7.5,甲基紫晶(MV)是氢酶催化放氢的最适人工电子载体,氧对氢酶催化活性有较大的抑制作用.The hydrogenase-containing crude extraction of Klebsiella oxytoca HP1 was firstly precipitated by 20% and 60% of ammonium sulfate solution in water,respectively.It was subsequently purified by three steps of column chromatography(DEAE-Sepharose F.F.,Sephacryl S-200 and TSK-DEAE).After three steps of purification,the soluble hydrogense of K.oxytoca HP1 was purified about 200 folds with a yield of 15.3% and a specific activity of 17.68 μmol H_2/min·mg prot.The catalytic property of soluble hydrogenase was studied.The optimal conditions for hydrogen production were obtained as follow: temperature 30℃,pH 7.5.Oxygen was an inhibitor for the hydrogen production of soluble hydrogenase.MV was the optimal electron carrier for the soluble hydrogenase′s activity.国家自然科学基金(30470395);; 厦门大学新世纪优秀人才支持计划(X07114)资
