EXPRESSION OF GENE ENCODING LIPASE (LIPA) FROM BACILLUS SUBT/LIS FS2

Abstract

The gene encoding an extracellular lipase from Bacillus subtilis FS2 was cloned using PCR technique. An open reading frame (ORF) of 543 bp encoding lipase A from genomic DNA of B. subtilis FS2 was cloned into vector pBlueScriptSK(+) forming pBlueLipA recombinant plasmid. The LipA gene obtained by digestion of pBlueLipA vector with BamHI and XhoI was inserted into pET22b( +) resulting in the recombinant vector pETLipA. The recombinant vector containing LipA gene was transformed into E. coli BL21 cells for expressing in LB medium. The recombinant protein of -24 kDa was expressed in E. coli BL21 cell after induction at 2 hours with 1 mM IPTG and the strongest expression was at 5 hours after induction with a protein concentration of 0.5 mglml. The recombinant protein containing 6 histidine (His) residues at C terminal with an isoelectric point (pI) 9.2 was purified through an affinity chromatography using 5 ml HiTrap column. The enzymatic activity of the purified recombinant protein was determined on an agar plate containing 0.1 % tributyrin. The enzyme has the specific activity of about 19,814 U/mg