Cloning and Expression of Gene Encoding Lipase from Local Isolate Bacillus cereus Isolated from Compost Jambangan Indonesia

Abstract

Bacillus cereus from Jambangan compost showed lipolytic activity and produce the thermostable lipase. Cloning and expression of lipase gene from the local bacteria was done to increase the enzyme production to support its use in many fields. In this research, the lipase gene was isolated from Bacillus cereus by PCR method using a pair of F-Lip and R-Lip primers, then cloned in E. coli using the pGemT vector and expressed with pCold II DNA vector. The amplification of lip gene by PCR could produce DNA fragments measuring 0.9 kb. The DNA fragment was then inserted into the pGemT cloning vector and resulted in pGemT-liprecombinant at 3.9 kb. The DNA fragment 3.9 kb represented a combination of pGemT size (3.0 kb) with lip gene (0.9 kb). The expression of the lipase gene in the E. coli BL21 (DE3) host was carried out with the pCold II DNA expression vector resulting in recombinant DNA at 5.2 kb. The 4.3 kb DNA fragment corresponds to the empty pCod II-DNA plasmid DNA, while the 0.9 kb fragment corresponds to lip gene. The production of recombinant lipase was carried out by cold shock technique at 15 oC when the culture reached OD 600 0.4-0.5 and was followed by induction of 0.1 mM IPTG. The results of SDS-PAGE analysis showed the presence of protein band 30 kDa at SDS PAGE electroforegram. The enzyme showed specific activity of as46.03 U/mg. The results indicated the lip gene encoding lipase fromBacillus cereus could be expressed well in the host of E. coli BL21 (DE3).