The NADPH-dependent cytoplasmic [NiFe]-hydrogenase (SHI) from the hyperthermophile Pyrococcus furiosus, which grows optimally near 100°C, is extremely thermostable and has many in vitro applications, including cofactor generation and hydrogen production. In particular, SHI is used in a cell-free synthetic pathway that contains more than a dozen other enzymes and produces three times more hydrogen (12 H₂/glucose) from sugars compared to cellular fermentations (4 H₂/glucose). We previously reported homologous over-expression and rapid purification of an affinity-tagged (9x-His) version of SHI, which is a heterotetrameric enzyme. However, about 30% of the enzyme that was purified contained an inactive trimeric form of SHI lacking the catalytic [NiFe]-containing subunit. Herein, we constructed a strain of P. furiosus that contained a second set of the eight genes involved in the maturation of the catalytic subunit and insertion of the [NiFe]-site, along with a second set of the four genes encoding the SHI structural subunits. This resulted in a 40% higher yield of the purified affinity-tagged enzyme and the content of the inactive trimeric form decreased to 5% of the total protein. These results bode well for the future production of active SHI for both basic and applied purposes.

中文翻译：

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通过增加成熟基因的表达来改善激烈热球菌NiFe氢化酶的生产

来自嗜热嗜热球菌的NADPH依赖性细胞质[NiFe]-加氢酶（SHI）最佳生长在100°C附近，具有极高的热稳定性，并具有许多体外应用，包括辅因子的产生和氢的产生。尤其是，SHI用于无细胞的合成途径，该途径包含十几种其他酶，并且与细胞发酵（4 H ₂）相比，糖产生的氢（12 H ₂ /葡萄糖）多三倍/葡萄糖）。我们先前报道了SHI（一种异四聚体酶）的亲和标记（9x-His）版本的同源过表达和快速纯化。但是，纯化的酶中约有30％含有SHI的无活性三聚体形式，而缺少催化的[NiFe]亚基。在这里，我们构建了一个P. furiosus菌株，该菌株包含参与催化亚基成熟和[NiFe]位点插入的八个基因的第二组，以及编码SHI结构的四个基因的第二组亚单位。这导致纯化的亲和标签酶的产率提高了40％，无活性三聚体形式的含量降低到总蛋白的5％。这些结果预示了未来用于基础和应用目的的活性SHI的良好前景。