title: Heterologous overexpression, purification and characterisation of an alcohol dehydrogenase (ADH2) from Halobacterium sp. NRC-1.
abstract: Replacement of chemical steps with biocatalytic ones is becoming increasingly more interesting due to the remarkable catalytic properties of enzymes, such as their wide range of substrate specificities and variety of chemo-, stereo- and regioselective reactions. This study presents characterisation of an alcohol dehydrogenase (ADH) from the halophilic archaeum Halobacterium sp. NRC-1 (HsADH2). A hexahistidine-tagged recombinant version of HsADH2 (His-HsADH2) was heterologously overexpressed in Haloferax volcanii. The enzyme was purified in one step by immobilised Ni-affinity chromatography. His-HsADH2 was halophilic and mildly thermophilic with optimal activity for ethanol oxidation at 4 M KCl around 60 °C and pH 10.0. The enzyme was extremely stable, retaining 80 % activity after 30 days. His-HsADH2 showed preference for NADP(H) but interestingly retained 60 % activity towards NADH. The enzyme displayed broad substrate specificity, with maximum activity obtained for 1-propanol. The enzyme also accepted secondary alcohols such as 2-butanol and even 1-phenylethanol. In the reductive reaction, working conditions for His-HsADH2 were optimised for acetaldehyde and found to be 4 M KCl and pH 6.0. His-HsADH2 displayed intrinsic organic solvent tolerance, which is highly relevant for biotechnological applications.
authors: Centre for Synthesis and Chemical Biology, School of Chemistry, University College Dublin, Belfield, Dublin 4, Ireland.
authors: Ann-Kathrin
authors: AK
authors: Liliensiek
authors: Jennifer
authors: J
authors: Cassidy
authors: Gabriele
authors: G
authors: Gucciardo
authors: Cliadhna
authors: C
authors: Whitely
authors: Francesca
authors: F
authors: Paradisi
mesh_terms: D000433:1-Propanol / Q000378:metabolism; D000426:Alcohol Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D000438:Alcohols / Q000378:metabolism; D019843:Archaeal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D001592:Benzyl Alcohols / Q000378:metabolism; D055162:Biocatalysis; D000440:Butanols / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004795:Enzyme Stability; D019766:Genes, Archaeal; D006217:Halobacterium / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D019622:Haloferax volcanii / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012997:Solvents; D013379:Substrate Specificity; D014169:Transformation, Bacterial
chemical_list: D000438:Alcohols; D019843:Archaeal Proteins; D001592:Benzyl Alcohols; D000440:Butanols; D011994:Recombinant Proteins; D012997:Solvents; C043958:2-butanol; D009249:NADP; D000433:1-Propanol; C002017:methylphenyl carbinol; D000426:Alcohol Dehydrogenase
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