A cold-adapted esterase from psychrotrophic Pseudoalteromas sp. strain 643A.
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.
Cieśliński
Hubert
H
Departament of Microbiology, Gdańsk University of Technology, ul. Narutowicza 11/12, 80-952, Gdansk, Poland.
Białkowska
Aneta M
AM
Długołecka
Anna
A
Daroch
Maurycy
M
Tkaczuk
Karolina L
KL
Kalinowska
Halina
H
Kur
Józef
J
Turkiewicz
Marianna
M
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002384:Catalysis / Q000187:drug effects; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003080:Cold Temperature; D003300:Copper / Q000494:pharmacology; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D033201:Euphausiacea / Q000382:microbiology; D005978:Glutathione / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D031161:Pseudoalteromonas / Q000201:enzymology*; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D002413:Cations, Divalent; D011994:Recombinant Proteins; D010664:Phenylmethylsulfonyl Fluoride; D008623:Mercaptoethanol; D003300:Copper; D004950:Esterases; D005978:Glutathione; D002118:Calcium; D004229:Dithiothreitol