Sequence analysis of pseudomonas fluorescens esterase Ⅲ gene and characterization of the gene product expressed in escherichia coli

Abstract

A gene (estC) coding for a new esterase (esterase III) of Pseudomonas fluorescens was cloned into Escherichia coli JM83. DNA sequencing revealed a single open reading frame with GTG as a translation initiation codon for esterase III. This was confirmed by N-terminal amino acid sequence analysis of the purified esterase protein. The promoter region and a potential Shine-Dalgarno sequence were followed by the coding sequence of the estC gene. The amino acid sequence deduced from the nucleotide sequence cotains the consensus active site sequence, G-X-S-X-G, of serine esterase. The esterase expressed in an E. coli clone was purified by ion-exchange chromatography and gel filtration. The native form of the enzyme consisted of subunits, each with a molecular 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 esterase.