Department of Synthetic Biology and Bioenergy, J. Craig Venter Institute, Rockville, Maryland 20850, USA.
Appl Environ Microbiol. 2011 Mar;77(6):1990-8. doi: 10.1128/AEM.01559-10. Epub 2011 Jan 21.
Hydrogenases are enzymes involved in the bioproduction of hydrogen, a clean alternative energy source whose combustion generates water as the only end product. In this article we identified and characterized a [NiFe] hydrogenase from the marine bacterium Alteromonas macleodii "deep ecotype" with unusual stability toward oxygen and high temperature. The A. macleodii hydrogenase (HynSL) can catalyze both H(2) evolution and H(2) uptake reactions. HynSL was expressed in A. macleodii under aerobic conditions and reached the maximum activity when the cells entered the late exponential phase. The higher level of hydrogenase activity was accompanied by a greater abundance of the HynSL protein in the late-log or stationary phase. The addition of nickel to the growth medium significantly enhanced the hydrogenase activity. Ni treatment affected the level of the protein, but not the mRNA, indicating that the effect of Ni was exerted at the posttranscriptional level. Hydrogenase activity was distributed ∼30% in the membrane fraction and ∼70% in the cytoplasmic fraction. Thus, HynSL appears to be loosely membrane-bound. Partially purified A. macleodii hydrogenase demonstrated extraordinary stability. It retained 84% of its activity after exposure to 80°C for 2 h. After exposure to air for 45 days at 4°C, it retained nearly 100% of its activity when assayed under anaerobic conditions. Its catalytic activity in the presence of O(2) was evaluated by the hydrogen-deuterium (H-D) exchange assay. In 1% O(2), 20.4% of its H-D exchange activity was retained. The great stability of HynSL makes it a potential candidate for biotechnological applications.
氢化酶是参与生物制氢的酶，氢气是一种清洁的替代能源，其燃烧生成水作为唯一的终产物。在本文中，我们鉴定并表征了一种来自海洋细菌 Alteromonas macleodii“深海型”的[NiFe]氢化酶，该酶对氧气和高温具有不寻常的稳定性。A. macleodii 氢化酶（HynSL）可以催化 H2 的产生和吸收反应。在好氧条件下，HynSL 在 A. macleodii 中表达，并在细胞进入指数晚期时达到最大活性。较高水平的氢化酶活性伴随着 HynSL 蛋白在对数晚期或静止期的丰度增加。在生长培养基中添加镍显著增强了氢化酶的活性。Ni 处理影响了蛋白的水平，但不影响 mRNA，表明 Ni 的作用发生在转录后水平。氢化酶活性分布在膜部分约 30%，细胞质部分约 70%。因此，HynSL 似乎是松散地与膜结合的。部分纯化的 A. macleodii 氢化酶表现出非凡的稳定性。在 80°C 下暴露 2 小时后，它保留了 84%的活性。在 4°C 下暴露于空气 45 天后，在厌氧条件下进行测定时，它保留了近 100%的活性。其在 O2 存在下的催化活性通过 H-D 交换测定来评估。在 1%的 O2 中，保留了其 H-D 交换活性的 20.4%。HynSL 的高稳定性使其成为生物技术应用的潜在候选者。
