产酸克雷伯氏菌氢酶分离提纯与特性的初步研究

Primary Studies on Purification and Properties of Soluble Hydrogenase from Klebsiella oxytoca HP1

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 17.68μmol H2/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 hydrogenasee＇s activity.