Molecular Cloning, Purification, and Characterization of a Cold-Adapted Esterase from Photobacterium sp. MA1-3

摘要

The gene encoding an esterase from Photobacterium sp. MA 1-3 was cloned in Escherichia coli using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (948 bp) corresponded to a protein of 315 amino acid residues with a molecular weight of 35 kDa and a pi of 6.06. The deduced protein showed 74% and 68% amino acid sequence identities with the putative esterases from Photobacterium profundum SS9 and Photobacterium damselae, respectively. Absence of a signal peptide indicatedthat it was a cell-bound protein. Sequence analysis showed that the protein contained the signature G-X-S-X-G included in most serine-esterases and lipases. The MAI-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboringthe gene were cultured at 18°C. The enzyme was a serine-esterase and was active against C2, C4, C8and Cl0 p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and 30°C, respectively. Relative activity remained up to 45%even at 5°C with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by Cd~(2+), Cu~(2+), Zn~(2+), and Hg~(2+) ions.