Ueno Y, Kurano N, Miyachi S
Marine Biotechnology Institute, Kamaishi Laboratories, Iwate, Japan.
FEBS Lett. 1999 Jan 25;443(2):144-8. doi: 10.1016/s0014-5793(98)01699-8.
Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485-fold, resulting in a specific activity for hydrogen evolution of 75.7 micromol/min/mg of protein at 25 degrees C, using reduced methyl viologen as an electron donor. The K(m) value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS-PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50 degrees C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K(m) value for ferredoxin was 0.68 microM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4 degrees C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.
从海洋绿藻滨海绿球藻中纯化得到的氢化酶，纯化倍数为1485倍，在25℃下，以还原型甲基紫精作为电子供体时，其产氢的比活性为75.7微摩尔/分钟/毫克蛋白质。甲基紫精的米氏常数（K(m)）值为0.5毫摩尔。通过非变性聚丙烯酰胺凝胶电泳（native PAGE）判断该酶的纯度。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）估计其分子量为55千道尔顿，通过凝胶过滤法估计为57千道尔顿。产氢的最适温度和pH值分别为50℃和7.5。部分纯化的氢化酶能催化从同一细胞中分离得到的铁氧化还原蛋白产氢，但不能催化烟酰胺腺嘌呤二核苷酸（NADH）或烟酰胺腺嘌呤二核苷酸磷酸（NADPH）产氢。铁氧化还原蛋白的米氏常数（K(m)）值为0.68微摩尔。该酶对氧气极其敏感，即使在4℃下，暴露于空气中几分钟后其活性就会丧失超过95%。通过酶解从氢化酶蛋白中获得了两个肽段，并测定了它们的氨基酸序列。未发现与其他已知氢化酶序列有明显的同源性。
