Cloning and expression of esterase gene of pseudomonas fluorescens and characterization of the gene product

Abstract

A gene (estA) coding for arylesterase (esterase I) of Pseudomonas fluorescens was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. From the plasmid harboring estA, a smaller plasmid pUE1251, was obtained by reducing the size of the inserted DNA from 9.8 to 1.2-kb. DNA sequencing showed that the open reading frame is comprised of 708 nucleotides which can potentially produce a precursor of 236 amino acids with a predicted molecular weight of 26,936. The G+C content of the coding sequence was found to be 62\%. The promoter-like structure (-10 and -35 regions) and a potential Shine-Dalgarno sequence are followed by the coding sequence of estA gene. Following the stop codon (TGA) a structure reminiscent of the Escherichi a coli rho-independent terminator is present. The arylesterase was purified from Escherichia coli JM83 harboring plasmid pUE1251. The enzyme expressed in an E. coli clone was mostly in the periplasmic space, released to the outside of the cell by osmotic shock and purified to homogeneity by QAE-Sephadex A-50 and DEAE-Sepharose columns. The amino acid sequence and the open reading frame deduced from the nucleotide sequence was confirmed by N-terminal amino acid sequence analysis of the purified enzyme protein. SDS-polyacrylamide gel electrophoresis of the purified arylesterase yields a single protein band with a relative molecular weight of 27,000. The native molecular weight was estimated to be 56,000 by gel filtration on Sephacryl S-200, indicating that the enzyme consists of two identical subunits. Orthorhombic and hexagonal crystal of arylesterase (esterase I) were obtained by the batch and vapor diffusion methods. Arylesterase is maximally active at pH 6.5 to 9.0, and the optimum temperature for this enzyme was 55 $^\circ$C. The enzyme exhibited maximum activity when phenylacetate was used as a substrate. The enzyme activity was not inhibited by disopropylfluorophosphate and paraxon. Co...