Cloning Nucleotide Sequence And Expression In Escherichia Coli Of A Lipase Gene From Geobacillus Stearothermophilus

Lipase(triacylglycerol acylhydrolases, EC 3.1.1.3), which are widely existed in animals, plants and microorganisms, catalyze both hydrolysis and synthesis of ester bonds of triglycerides. Lipase are thus of particular importance in detergents, food ingredients, lether and paper industry and many other industrial applications. Recently, basic properties and industrial applications of thermostable lipase from mesophiles and thermophiles attract a lot of interests, one of these thermophile is newly named bacteria Geobacillus stearothermophilus in 2001. Although thermostable lipases have great advantages, they are normally produced at low levels. Most of up-to-date studies focus on the cloning and expression of lipase gene in different expression systems by gene engineering methods.In this thesis, Geobacillus stearothermophilus was cultured at 65℃ in the culture containing tryptone, yeast extract, meat extract and NaCl at pH 7.0. The genome DNA was used as template for PCR and Pfu as DNA polymerase. 1.3kb band generated by PCR was cloned into pMD18-T Simple Vector.After the correct sequence was confirmed, plasmid pET28c(+) and TA clone were digested by BarnH I and Nde I respectively and ligated by T4 DNA ligase to construct the recombinant plasmid pET28c(+)-lipase with 6590bp.This plasmid was amplified in DH5α.Then recombinant plasmid was transformed into E.coli BL21(DE3) to express lipase. Expression was confirmed by SDS-PAGE and lipase assay. Optimized Expression condition of lipase were: OD₆₀₀ at 1.0,28℃,6h after induced by 0.5mmol/L IPTG. Lipase specific activity was 1.88U/mg when olive oil as substrate.In addition, properties of lipase produced by recombinant E.coli were studied. Recombinant lipase reached the highest activity in pH 8.5 Tris-HCl(0.05mol/L) at 55℃. The halflife of lipase at 55℃ was about 8hous. 10mM Zn²⁺,Fe²⁺ caused almost complete block of lipase activity. Lipase activity was significantly decreased by 10mM PMSF after 30min incubation.