Nucleotide sequence of a novel arylesterase gene from Vibro mimicus and characterization of the enzyme expressed in Escherichia coli

Abstract

A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequence determination reveals that the esterase gene has an open reading frame of 600 nucleotides which encodes a protein of M(r) 22,300. The deduced amino acid sequence contain a pentapeptide GDSLS (residues 27-31), which was also found in the phospholipid-cholesterol acyltransferase from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cysteine in the cloned gene abolished the esterase activity in the tributyrin plate assay. On the other hand, the activity was not lost when Ser-31 was changed to alanine. The cloned gene was expressed in Escherichia coli, and the protein purified by a four-step procedure. The purified protein migrated on SDS/PAGE as a single band with an apparent M(r) of 22,100. This enzyme favoured the hydrolysis of several arylesters and was classified as an arylesterase (EC 3.1.1.2). N-Terminal analysis showed that Ser-20 was the first amino acid of the mature secreted protein, suggesting that the N-terminal 19 hydrophobic amino acids served as a signal peptide.