Expression, Purification, and Enzyme Activity Assay of a Recombinant Aldehyde Dehydrogenase from , Using an Host.

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.