NUCLEOTIDE-SEQUENCE OF A NOVEL ARYLESTERASE GENE FROM VIBRIO-MIMICUS AND CHARACTERIZATION OF THE ENZYME EXPRESSED IN ESCHERICHIA-COLI

A gene coding for an arylesterase of Vibrio mimicus was cloned. Sequen ce determination reveals that the esterase gene has an open reading fr ame of 600 nucleotides which encodes a protein of M(r) 22 300. The ded uced amino acid sequence contains a pentapeptide GDSLS (residues 27-31 ), which was also found in the phospholipid-cholesterol acyltransferas e from Aeromonas hydrophila. Substitution of Ser-29 by alanine or cyst eine in the cloned gene abolished the esterase activity in the tributy rin plate assay. On the other hand, the activity was not lost when Ser -31 was changed to alanine. The cloned gene was expressed in Escherich ia coli, and the protein purified by a four-step procedure. The purifi ed 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 analysi s showed that Ser-20 was the first amino acid of the mature secreted p rotein, suggesting that the N-terminal 19 hydrophobic amino acids serv ed as a signal peptide.