A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A
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A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasiasuperba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichiacoli TOP10F’, and screening on tributyrin-containing agar plates led to the isolation of esterase gene. The esterase gene (estA, 621 bp) encoded a protein (EstA) of 207 amino acid residues with molecular mass of 23,036 Da. Analysis of the amino acid sequence of EstA suggests that it is a member of the GDSL-lipolytic enzymes family. The purification and characterization of native EstA esterase were performed. The enzyme displayed 20–50% of maximum activity at 0–20°C. The optimal temperature for EstA was 35°C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C4 and C10) fatty acids, and exhibited no activity for long-chain fatty acid esters like that of palmitate and stearate. EstA was strongly inhibited by phenylmethylsulfonyl fluoride, 2–mercaptoethanol, dithiothreitol and glutathione. Addition of selected divalent ions e.g. Mg2+, Co2+ and Cu2+ led to the reduction of enzymatic activity and the enzyme was slightly activated (∼30%) by Ca2+ ions.
KeywordsCold-adapted enzyme Psychrotolerant bacterium Esterases/lipases GDSL esterase Cloning
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