Cloning, Expression Of Lipase Gene From Serratia Marcescens In Bacillus Subtilis And Its Fermentation Optimization

Lipase is a kind of ester bond hydrolase which can decompose natural oils and fats intofatty acids, glycerol and monoglyceride or diglyceride. Recently, with the rapid developmentof enzyme technology, bio-lipase has been widely applied in many fields such as food,medicine, detergent, oil processing, bio-diesel. Lipase from Serratia marcescens has a greatpotential in the synthesis of chiral drugs and biodiesel for its high stereoselectivity and goodtolerance of methanol, but the opportunistic pathogenic of S. marcescens limits itsapplications. So heterologous expression of the S. marcescens lipase gene in the secure host isalways an important direction of the research in this field.1) Four lipase-producing strains were screened from the soil samples retrieved fromdifferent sampling sites, their lipase enzyme activitives were assayed, and one’s of the fourwas significantly higher than that of the other three. The strain was identified to be S.marcescens and named S. marcescens JNS3-9after physiological and biochemical and16SrDNA identification. The lipase enzyme activity of JNS3-9reached to8.57U/mL afterpreliminary fermentation experiment.2) The lipA gene from S. marcescens JNS3-9was obtained by PCR. The recombinantplasmid pET-28a-lipA was constructed and then transformed into Escherichia coli BL21toconstruct the recombinant strain E. coli BL21/pET28a-lipA. The successfully expressed lipasewas analyzed by SDS-PAGE. The activities of the recombinant enzyme in E. coliBL21/pET28a-lipA were assayed.3) The recombinant plasmid pMA5-lipA was constructed and then transformed intoBacillus subtilis168to construct the recombinant strain B. subtilis168/pMA5-lipA. Theactivity of the recombinant enzyme in B. subtilis168/pMA5-lipA were33.24U/mL, whichwas about4-fold that of the wild type in S. marcescens JNS3-9. The recombinant lipase waspurified by Ni-NTA, and the enzyme properties including optimum temperature,temperaturestability, optimum pH, and pH stability, the influence of metal ions on the enzyme and organicsolvent tolerance were studied.4) The fermentation medium composition and culture conditions of B. subtilis168/pMA5-lipA were optimized in shake flask level. The lipase yield reached to98.6U/mLin the optimal fermentation medium and culture conditions, which was3times of theprevious.