Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West 7th Avenue, Tianjin Airport Economic Area, Tianjin, 300308, China.
College of Life Sciences, Henan Agricultural University, 95 Wenhua Road, Zhengzhou, 450002, China.
Biotechnol J. 2019 Apr;14(4):e1800301. doi: 10.1002/biot.201800301. Epub 2018 Oct 26.
Soluble hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus is a heterotetrameric [NiFe] hydrogenase that catalyzes the reversible reduction of protons by NADPH into hydrogen gas (H ). Here, the authors expressed the four αβγδ subunits of SHI encoded by one gene cluster in another hyperthermophilic archaeon, Thermococcus kodakarensis KOD1, which uses its hydrogenase maturation apparatus without the coexpression of native P. furiosus hydrogenase endopeptidases (maturation proteases). The SHI overexpression of T. kodakarensis resulted in more than 1200-fold enhancement in the hydrogenase activity of the cell lysate compared to that of the host strain with an empty vector. An active, purified 12-His tagged recombinant SHI (rSHI) is obtained by one-step affinity adsorption on nickel-charged resin. Size-exclusion chromatography show that purified rSHI is heterotetrameric and has a molecular mass of 150 kDa. The purified rSHI has a half-life of 70 h at 80 °C. This rSHI is used to design a novel in vitro synthetic enzymatic biosystem to convert pyruvate and H gas into lactate in a theoretical yield, whereas rSHI is used for NADPH regeneration; an FMN-containing diaphorase (DI) is used to match NADP-preferred SHI and NAD-preferred lactate dehydrogenase (LDH). This study provides a cost-efficient method to obtain hyperthermostable hydrogenases, which can be used in in vitro synthetic enzymatic biosystems for cofactor regeneration and hydrogen production.
来自嗜热古菌 Pyrococcus furiosus 的可溶性氢化酶 I（SHI）是一种异源四聚体 [NiFe]氢化酶，可催化质子与 NADPH 的可逆还原反应生成氢气（H ）。在这里，作者在另一种嗜热古菌 Thermococcus kodakarensis KOD1 中表达了由一个基因簇编码的 SHI 的四个 αβγδ 亚基，该基因簇利用其氢化酶成熟装置，而无需共表达天然 P. furiosus 氢化酶内切蛋白酶（成熟蛋白酶）。与含有空载体的宿主菌株相比，Thermococcus kodakarensis 的 SHI 过表达使细胞裂解物中的氢化酶活性增强了 1200 多倍。通过一步亲和吸附在镍 charged 树脂上获得了活性、纯化的 12-His 标记的重组 SHI（rSHI）。分子筛层析表明，纯化的 rSHI 是异源四聚体，分子量为 150 kDa。纯化的 rSHI 在 80°C 下半衰期为 70 h。该 rSHI 用于设计一种新型的体外合成酶生物系统，以理论产率将丙酮酸和 H 气体转化为乳酸，而 rSHI 用于 NADPH 再生；含有 FMN 的黄递酶（DI）用于匹配 NADP 偏好的 SHI 和 NAD 偏好的乳酸脱氢酶（LDH）。该研究提供了一种经济高效的方法来获得耐热氢化酶，可用于体外合成酶生物系统中的辅因子再生和氢气生产。
