Gene cloning and characterization of a cold-adapted esterase from Acinetobacter venetianus V28.
Acinetobacter venetians V28 was isolated from the intestine of righteye flounder, Poecilopsetta plinthus caught in Vietnam seawater, and the esterase gene was cloned using a shotgun method. The amino acid sequence deduced from the nucleotide sequence (1,017 bp) corresponded to a protein of 338 amino acid residues with a molecular weight of 37,186. The esterase had 87% and 72% identities with the lipases of A. junii SH205 and A. calcoaceticus RUH2202, respectively. The esterase contained a putative leader sequence, as well as the conserved catalytic triad (Ser, His, Asp), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein from the strain V28 was produced in both a soluble and an insoluble form when the Escherichia coli cells harboring the gene were cultured at 18 degrees C. The maximal activity of the purified enzyme was observed at a temperature of 40 degrees C and pH 9.0 using p-NP-caprylate as substrate; however, relative activity still reached to 70% even at 5 degrees C with an activation energy of 3.36 kcal/mol, which indicated that it was a cold-adapted enzyme. The enzyme was a nonmetalloprotein and was active against p-nitrophenyl esters of C4, C8, and C14. Remarkably, this enzyme retained much of its activity in the presence of commercial detergents and organic solvents. This cold-adapted esterase will be applicable as catalysts for reaction in the presence of organic solvents and detergents.
Biotechnology Research Division, National Fisheries Research and Development Institute, Busan, Korea. yokim@nfrdi.go.kr
Young-Ok
YO
Kim
Yu Li
YL
Heo
Hyung-Kwoun
HK
Kim
Bo-Hye
BH
Nam
Hee Jeong
HJ
Kong
Dong-Gyun
DG
Kim
Woo-Jin
WJ
Kim
Bong-Seok
BS
Kim
Young-Ju
YJ
Jee
Sang-Jun
SJ
Lee
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D000438:Alcohols; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D057773:Cold Shock Proteins and Peptides / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003902:Detergents; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D004950:Esterases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005432:Flounder / Q000382:microbiology; D006863:Hydrogen-Ion Concentration; D007422:Intestines / Q000382:microbiology; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D013696:Temperature; D013816:Thermodynamics
D000438:Alcohols; D057773:Cold Shock Proteins and Peptides; D003902:Detergents; D011994:Recombinant Proteins; D004950:Esterases