title: A novel cold-adapted esterase from Salinisphaera sp. P7-4: gene cloning, overproduction, and characterization.
abstract: Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.
authors.affiliation: Biotechnology Research Division, National Fisheries Research and Development Institute, 408-1 Sirang-Ri, Busan, South Korea. yokim@nfrdi.go.kr
authors.forename: Young-Ok
authors.initials: YO
authors.lastname: Kim
authors.forename: In-Suk
authors.initials: IS
authors.lastname: Park
authors.forename: Hyung-Kwou
authors.initials: HK
authors.lastname: Kim
authors.forename: Bo-Hye
authors.initials: BH
authors.lastname: Nam
authors.forename: Hee
authors.initials: H
authors.lastname: Jeong Kong
authors.forename: Woo-Jin
authors.initials: WJ
authors.lastname: Kim
authors.forename: Dong-Gyun
authors.initials: DG
authors.lastname: Kim
authors.forename: Kyung-Kil
authors.initials: KK
authors.lastname: Kim
authors.forename: Sang-Jun
authors.initials: SJ
authors.lastname: Lee
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D003080:Cold Temperature; D020118:Cupriavidus necator / Q000201:enzymology / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D004950:Esterases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D020563:Gammaproteobacteria / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010140:Pacific Ocean; D010473:Perciformes / Q000382:microbiology; D012241:Rhodopseudomonas / Q000201:enzymology / Q000235:genetics; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
chemical_list: D004791:Enzyme Inhibitors; D008670:Metals; D004950:Esterases