CLONING OF PSEUDOMONAS-FLUORESCENS CARBOXYLESTERASE GENE AND CHARACTERIZATION OF ITS PRODUCT EXPRESSED IN ESCHERICHIA-COLI

A gene (estC) coding for an esterase (esterase III) of Pseudomonas flu orescens was cloned into Escherichia coli JM83. DNA sequencing showed a single open reading frame with GTG as a translation initiation codon for esterase III. This was confirmed by N-terminal amino acid sequenc e analysis of the purified esterase III protein from an E. coli clone. The promoter sequence and a potential Shine-Dalgarno sequence were fo llowed by the coding sequence of the estC gene. The amino acid sequenc e deduced from the nucleotide sequence contains the consensus active s ite sequence, G-X-S-X-G, of serine esterase. The esterase III expresse d in an E. coli clone was purified by ion-exchange chromatography and gel filtration. The native form of the enzyme was a monomer with a mol ecular weight of 41,000. The results of substrate specificity and the inhibitor studies suggest that this enzyme is a carboxylesterase (EC 3 .1.1.1) and a serine residue is present at the active site of the enzy me.