H2 Production from Methyl Viologen–Dependent Hydrogenase Activity Monitored by Gas Chromatography

N. Kosem
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Abstract

Bio-hydrogen production is an eco-friendly alternative to commercial H2 production, taking advantage of natural systems. Microbial hydrogenases play a main role in biological mechanisms, catalyzing proton reduction to molecular hydrogen (H2) formation under ambient conditions. Direct determination is an important approach to screen bacteria with active hydrogenase and accurately quantify the amount of H2 production. Here, we present a detailed protocol for determining hydrogenase activity based on H2 production using methyl viologen (MV2+) as an artificial reductant, directly monitored by gas chromatography. Recombinant Escherichia coli is used as a hydrogenase-enriched model in this study. Even so, this protocol can be applied to determine hydrogenase activity in all biological samples. Key features • This protocol is optimized for a wide variety of biological samples; both purified hydrogenase (in vitro) and intracellular hydrogenase (in vivo) systems. • Direct, quantitative, and accurate method to detect the amount of H2 by gas chromatography with reproducibility. • Requires only 2 h to complete and allows testing various conditions simultaneously. • Kinetic plot of H2 production allows to analyze kinetic parameters and estimate the efficiency of hydrogenase from different organisms.
通过气相色谱法监测依赖甲基维奥根的氢化酶活性产生的 H2
生物制氢是商业制氢的环保替代品,利用了自然系统的优势。微生物氢化酶在生物机制中起着重要作用,在环境条件下催化质子还原成分子氢(H2)。直接测定法是筛选具有活性氢化酶的细菌和准确定量产氢量的重要方法。在这里,我们提出了一种详细的方案,以甲基紫素(MV2+)作为人工还原剂,通过气相色谱法直接监测氢气的产生,以确定氢化酶的活性。本研究采用重组大肠杆菌作为氢化酶富集模型。即使如此,该方案可适用于确定氢化酶活性在所有的生物样品。主要特点•该协议是各种生物样品的优化;纯化氢化酶(体外)和细胞内氢化酶(体内)系统。•直接,定量,准确的方法,以气相色谱法检测H2的量,具有重复性。•只需2小时即可完成,并允许同时测试各种条件。•H2生产的动力学图允许分析动力学参数并估计来自不同生物的氢化酶的效率。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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