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首页> 外文期刊>Protein Expression and Purification >Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana
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Purification and enzymatic characterization of alcohol dehydrogenase from Arabidopsis thaliana

机译：拟南芥醇脱氢酶的纯化及酶学性质

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摘要

Alcohol dehydrogenases (ADH) catalyze the interconversion between alcohols and aldehydes with the reduction of nicotinamide adenine dinucleotide (NAD ~+) to NADH. In this study, for the first time we report an over-expression and purification strategy for the Arabidosis thaliana ADH (AtADH), and characterize its enzymatic properties. AtADH was expressed in an Escherichia coli system, the polyhistidine-tag was removed after the recombinant AtADH protein was purified by metal chelating affinity chromatography. Activity assays demonstrated that AtADH has distinct enzymatic properties when compared with many well-known ADHs. It held peak activity at pH 10.5 and showed broad substrate selectivity for primary and secondary alcohols. The kinetic K _m parameters for both ethanol and coenzyme were in the order of mM. This relative low affinity may reflect the need of the plant to maintain a supply of NAD~+ in nature. Different from yeast ADH, AtADH showed almost the same activity for short straight chain alcohols and reduced activity for secondary alcohols. This broad spectrum in alcohol selection and the observed higher catalytic activity (high V_(max) (EtOH)) may result from the requirement of the single enzyme to accommodate many substrates.
机译：醇脱氢酶（ADH）催化烟酰胺之间的相互转化，将烟酰胺腺嘌呤二核苷酸（NAD〜+）还原为NADH。在这项研究中，我们首次报道了拟南芥ADH（AtADH）的过表达和纯化策略，并表征了其酶促性质。 AtADH在大肠杆菌系统中表达，通过金属螯合亲和层析纯化重组AtADH蛋白后，去除多组氨酸标签。活性分析表明，与许多众所周知的ADH相比，AtADH具有独特的酶促性质。它在pH 10.5时保持峰值活性，并显示出对伯醇和仲醇的广泛底物选择性。乙醇和辅酶的动力学K_m参数约为mM。这种相对较低的亲和力可能反映了植物维持自然界中NAD +的需求。与酵母ADH不同，AtADH对短直链醇的活性几乎相同，而对仲醇的活性却降低。酒精选择的广泛范围和观察到的更高的催化活性（高V_（max）（EtOH））可能是由于需要单一酶来容纳许多底物而导致的。

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