CLONING AND NUCLEOTIDE SEQUENCING OF A LIPASE GENE FROM BACILLUS-SUBTILIS WRRL-B558

A lipase gene from B. subtilis WRRL-B558 was cloned in Escherichia col i JM109 using pBluescript as a vector plasmid. Two methods were combin ed to screen for the lipase-producing clone. The first was done by ove rlaying the screening plates with beta-naphthylacetate and Fast Blue B E dye. Positive clones were then confirmed by a second method using 1% (v/v) tributyrin agar plates. Positive clones which formed clear zone s on the tributyrin agar plates were selected and analysed by restrict ion mapping. Southern blot hybridization and deletion studies to locat e the lipase gene on a 2.2 kb HindIII fragment insert. A subclone harb ouring a plasmid with a 0.9 kb DNA fragment between the HindlII and Ec oRI sites that still exhibited lipase activity was used for sequencing . The nucleotide sequence showed a single open reading frame which con tained 636 nucleotides (212 deduced amino acids). A conserved pentapep tide postulated to be the catalytic site was Ala-X-Ser-X-Gly instead o f Gly-X-Ser-X-Gly. The deduced protein was found to have a molecular w eight of 21 kDa which was similar to that obtained from the recombinan t plasmid as determined by SDS-PAGE. Expression of the Bacillus lipase gene was found to be high in recombinant E. coli.