A cold-adapted esterase from psychrotrophic <Emphasis Type="Italic">Pseudoalteromas</Emphasis> sp. strain 643A |
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Authors: | Hubert Cie?liński Aneta M Bia?kowska Anna D?ugo??cka Maurycy Daroch Karolina L Tkaczuk Halina Kalinowska Józef Kur Marianna Turkiewicz |
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Institution: | Departament of Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-952, Gdansk, Poland. |
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Abstract: | A psychrotrophic bacterium producing a cold-adapted esterase upon growth at low temperatures was isolated from the alimentary tract of Antarctic krill Euphasia superba Dana, and classified as Pseudoalteromonas sp. strain 643A. A genomic DNA library of strain 643A was introduced into Escherichia coli 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 degrees C. The optimal temperature for EstA was 35 degrees C. EstA was stable between pH 9 and 11.5. The enzyme showed activity for esters of short- to medium-chain (C(4) and C(10)) 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. Mg(2+), Co(2+) and Cu(2+) led to the reduction of enzymatic activity and the enzyme was slightly activated ( approximately 30%) by Ca(2+) ions. |
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Keywords: | Cold-adapted enzyme Psychrotolerant bacterium Esterases/lipases GDSL esterase Cloning |
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