International Institute for Carbon-Neutral Energy Research, Kyushu University, Nishi-ku, Fukuoka, Japan.
J Biosci Bioeng. 2013 Apr;115(4):366-71. doi: 10.1016/j.jbiosc.2012.10.011. Epub 2012 Nov 30.
[NiFeSe]hydrogenases are promising biocatalysts in H2-based technology due to their high catalytic activity and O2-stability. Here, we report purification and characterization of a new membrane-associated [NiFeSe]hydrogenase from Desulfovibrio vulgaris Miyazaki F ([NiFeSe]DvMF). The [NiFeSe]DvMF was composed of two subunits, corresponding to a large subunit of 58.3 kDa and a small subunit of 29.3 kDa determined by SDS-PAGE. Unlike conventional [NiFeSe]hydrogenases having catalytic bias toward H2-production, the [NiFeSe]DvMF showed 11-fold higher specific activity of H2-oxidation (2444 U/mg) than that of H2-production (217 U/mg). At the optimal reaction temperature of the enzyme (65°C), the specific activity of H2-oxidation could reach up to 21,553 U/mg. Amperometric assays of the [NiFeSe]DvMF clearly indicated that the enzyme had a remarkable O2-stability. According to the amino acid sequence alignment, the conserved cysteine residue at position 281 in medial cluster of other [NiFeSe]hydrogenases was specifically replaced by a serine residue (Ser281) in the [NiFeSe]DvMF. These results indicate that the [NiFeSe]DvMF can play as a new H2-oxidizing and O2-stable biocatalyst, along with providing helpful insights into the structure-function relationship of [NiFeSe]hydrogenases.
[NiFeSe]氢化酶由于其高催化活性和对 O2 的稳定性，是基于 H2 的技术中有前途的生物催化剂。在这里，我们报告了一种来自脱硫弧菌 Miyazaki F 的新型膜结合 [NiFeSe]氢化酶（[NiFeSe]DvMF）的纯化和表征。[NiFeSe]DvMF 由两个亚基组成，通过 SDS-PAGE 确定其大亚基为 58.3 kDa，小亚基为 29.3 kDa。与具有产氢催化偏向的传统 [NiFeSe]氢化酶不同，[NiFeSe]DvMF 对 H2 氧化的比活性（2444 U/mg）比 H2 产生的比活性（217 U/mg）高 11 倍。在酶的最佳反应温度（65°C）下，H2 氧化的比活性可高达 21,553 U/mg。[NiFeSe]DvMF 的安培测定清楚地表明，该酶具有显著的 O2 稳定性。根据氨基酸序列比对，其他 [NiFeSe]氢化酶中中簇的保守半胱氨酸残基 281 被[NiFeSe]DvMF 中的丝氨酸残基（Ser281）特异性取代。这些结果表明，[NiFeSe]DvMF 可以作为一种新的 H2 氧化和 O2 稳定的生物催化剂，同时为 [NiFeSe]氢化酶的结构-功能关系提供了有帮助的见解。
