Cloning and sequence analysis of a lipase gene from an unidentified bacterial species

Abstract

Lipase catalyzes the hydrolysis of long chain fatty acid ester to glycerol and fatty acids. The lipase gene was transferred from an unidentified bacterial species to $\mbox{\underline{Escherichia}}$ $\mbox{\underline{coli}}$ JM83 by a molecular cloning using pUC19 as a vector. The lipase from an unidentified bacterial species was characterized. It was maximally active at pH 8 to 9, and its optimum temperature was 40$^\circ$C. The chromosomal DNA was isolated from an unidentified bacterial species and partially digested with Sau3AI. The pUC19 vector was digested with BamHI and dephosphorylated with Calf intestinal alkaline phosphatase. The digested chromosomal DNA fragments and linearized pUC19 vector were ligated, and the resulting recombinant plasmids were transformed to $\mbox{\underline{E}}$. $\mbox{\underline{coli}}$ JM83. The first screening was carried out on the agar plate containing tributyrin to esterase or lipase activities. The second screening was performed on the agar plate containing olive oil and rhodamine-B to select the lipase activity. The recombinant plasmid isolated from the clone exhibiting lipase activity was designated as pUL9. pUL9 was linearized and digested with exonuclease Bal31 to reduce the size of the insert DNA and self ligated. The resulting recombinant plasmid was designated as pUL920. The nucleotide sequence of pUL920 was analyzed.