Cloning and overexpression of a thermostable lipase gene from pseudomonas fluorescens in escherichia coli

Abstract

A gene coding for lipase of $\underline{\mbox{Pseudomonas}}$ $\underline{\mbox{fluorescens}}$ SIK W1 was cloned into Escherichia coli JM83 by inserting Sau3AI-generated DNA fragments into the BamHI site of pUC19. Twenty one colonies exhibiting esterase activity on the tributyrin agar plate were isolated by screening the constructed $\underline{\mbox{Pseudomonas}}$ $\underline{\mbox{fluorescens}}$ genomic library. Only one out of the esterase positive 21 colonies exhibited lipase activity on the agar plate containing olive oil and rhodamine-B. The lipase gene was contained in a 10.3 kb chromosomal DNA fragment and the size of the chromosomal insert was reduced to 1.6 kb through a sequential subclonings. The complete nucleotide sequence of the lipase gene was determined. The lipase gene consists of an open reading frame, 1347 bp long, commencing ATG start codon encoding a polypetide of 449 amino acid residues and a TGA stop codon. 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 lipase. The G+C content of the lipase gene was found to be 62.5\%. Comparison of the lipase amino acid sequence with those form another organisms sequenced to date showed the presence of the short homologous region Gly-His-Ser-Leu-Gly. The lipase gene was cloned into expression vector, pTTQ19 and overexpressed in various $\underline{\mbox{Escherichia}}$ $\underline{\mbox{coli}}$ strains. The expression levels of the lipase gene under the control of tac promoter were strain specific, and among the strains tested, $\underline{\mbox{Escherichia}}$ $\underline{\mbox{coli}}$ BL21 was a good candidate as a host for the overproduction of the lipase. The lipase produced in Escherichia coli was exclusively located within the cytoplasm and existed as an biologically inactive protein aggregates, termed inclusion bodies. The amounts of protein produced by Escherichia coli BL21 were estimated to be m...