Cloning and nucleotide sequencing of a lipase gene from Bacillus subtilis WRRL-B558

• Main Authors: B. W. Thanomsub, C. Boonchird, V. Meevootisom
• Other Authors: Mahidol University
• Format: Article
• Published: 2018
• Subjects: Biochemistry, Genetics and Molecular Biology; Immunology and Microbiology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/17548

A lipase gene from B. subtilis WRRL-B558 was cloned in Escherichia coli JM109 using pBluescript as a vector plasmid. Two methods were combined to screen for the lipase-producing clone. The first was done by overlaying the screening plates with β-naphthylacetate and Fast Blue BB dye. Positive clones were then confirmed by a second method using 1% (v/v) tributyrin agar plates. Positive clones which formed clear zones on the tributyrin agar plates were selected and analysed by restriction mapping, Southern blot hybridization and deletion studies to locate the lipase gene on a 2.2 kb HindIII fragment insert. A subclone harbouring a plasmid with a 0.9 kb DNA fragment between the HindIII and EcoRI sites that still exhibited lipase activity was used for sequencing. The nucleotide sequence showed a single open reading frame which contained 636 nucleotides (212 deduced amine acids). A conserved pentapeptide postulated to be the catalytic site was Ala-X-Ser- X-Gly instead of Gly-X-Ser-X-Gly. The deduced protein was found to have a molecular weight of 21 kDa which was similar to that obtained from the recombinant plasmid as determined by SDS-PAGE. Expression of the Bacillus lipase gene was found to be high in recombinant E. coli.