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

Abstract

The gene encoding an esterase from Photobacterium sp. MA1-3 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 indicated that 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 MA1-3 esterase was produced in both soluble and insoluble forms when E. coli cells harboring the gene were cultured at <TEX>$18^{\circ}C$</TEX>. The enzyme was a serine-esterase and was active against <TEX>$C_2$</TEX>, <TEX>$C_4$</TEX>, <TEX>$C_8$</TEX> and <TEX>$C_{10}$</TEX> p-nitrophenyl esters. The optimum pH and temperature for enzyme activity were pH 8.0 and <TEX>$30^{\circ}C$</TEX>, respectively. Relative activity remained up to 45% even at <TEX>$5^{\circ}C$</TEX> with an activation energy of 7.69 kcal/mol, which indicated that it was a cold-adapted enzyme. Enzyme activity was inhibited by <TEX>$Cd^{2+}$</TEX>, <TEX>$Cu^{2+}$</TEX>, <TEX>$Zn^{2+}$</TEX>, and <TEX>$Hg^{2+}$</TEX> ions.