Shortall Kim, Magner Edmond, Soulimane Tewfik
Department of Chemical Sciences, Bernal Institute, University of Limerick, V94 T9PX, Limerick, Ireland.
Bio Protoc. 2022 May 5;12(9):e4401. doi: 10.21769/BioProtoc.4401.
Based on previous in-depth characterisation, aldehyde dehydrogenases (ALDH) are a diverse superfamily of enzymes, in terms of both structure and function, present in all kingdoms of life. They catalyse the oxidation of an aldehyde to carboxylic acid using the cofactor nicotinamide adenine dinucleotide (phosphate) (NAD(P)), and are often not substrate-specific, but rather have a broad range of associated biological functions, including detoxification and biosynthesis. We studied the structure of ALDH from , as well as performed its biochemical characterisation. This allowed for insight into its potential substrates and biological roles. In this protocol, we describe ALDH heterologous expression in , purification, and activity assay (based on Shortall , 2021 ). ALDH was first copurified as a contaminant during caa-type cytochrome oxidase isolation from . This recombinant production system was employed for structural and biochemical analysis of wild-type and mutants, and proved efficient, yielding approximately 15-20 mg/L ALDH. For purification of the thermophilic his-tagged ALDH, heat treatment, immobilized metal affinity chromatography (IMAC), and gel filtration chromatography were used. The enzyme activity assay was performed via UV-Vis spectrophotometry, monitoring the production of reduced nicotinamide adenine dinucleotide (NADH). Flow chart outlining the steps in ALDH expression and purification, highlighting the approximate time required for each step.
基于先前的深入表征，醛脱氢酶（ALDH）是一类结构和功能多样的酶超家族，存在于所有生命王国中。它们利用辅因子烟酰胺腺嘌呤二核苷酸（磷酸）（NAD(P)）催化醛氧化为羧酸，通常不具有底物特异性，而是具有广泛的相关生物学功能，包括解毒和生物合成。我们研究了来自[具体来源未提及]的ALDH的结构，并对其进行了生化表征。这有助于深入了解其潜在底物和生物学作用。在本方案中，我们描述了ALDH在[具体宿主未提及]中的异源表达、纯化和活性测定（基于Shortall，2021）。ALDH最初是在从[具体来源未提及]分离caa型细胞色素氧化酶时作为污染物共纯化得到的。该重组生产系统用于野生型和突变体的结构和生化分析，并证明是有效的，可产生约15 - 20 mg/L的ALDH。为了纯化嗜热组氨酸标签的ALDH，使用了热处理、固定化金属亲和色谱（IMAC）和凝胶过滤色谱。酶活性测定通过紫外可见分光光度法进行，监测还原型烟酰胺腺嘌呤二核苷酸（NADH）的产生。概述ALDH表达和纯化步骤的流程图，突出了每个步骤所需的大致时间。
