GENE CLONING, PURIFICATION, AND CHARACTERIZATION OF A COLD‐ADAPTED LIPASE FROM ACINETOBACTER SP. V28‐28

Abstract

A bacterial strain that produced extracellular lipase was isolated from seawater and identified as Acinetobacter sp. V28‐28. In the present study, the corresponding gene was cloned using 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 lipase had 87 and 72% identities with the lipases of Acinetobacter junii SH205 and Acinetobacter calcoaceticus RUH2202, respectively. The lipase 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 V28‐28 was produced in both a soluble and insoluble form when the Escherichia coli cells harboring the gene were cultured at 18?. The optimum pH and temperature for the enzyme activity was pH 9.0 and 40?, respectively. 50% of the activity observed at 40? was still remaining at 5?. The stability of the purified enzyme at low temperatures indicates that it is a cold‐adapted enzyme. The enzyme showed the highest activity toward the ?‐nitrophenyl ester of caprylate (C8) and metal ion such as Zn2+ inhibited enzymatic activity. Partial characterization indicated that the lipase from Acinetobacter sp. V28‐28 is a new cold‐adapted enzyme.