Studies On Isolation Of Organic Solvent Resistant Strains With High Lipase Activity And Purification Of The Enzyme

Lipases(EC3.1.1.3) are glycerol ester hydrolases, they can catalyze the hydrolysis of triacyglycerols into free fatty acids,partial acylglycerols and glycerol. Its catalytic effect has the regional selective and the precise stereoselective. It is not only in the out-of-phase system(oil-water interface), which play a role, in addition, they also remain active in a variety of organic solvents. In recent years, lipase applications are beyond the oil- water interface on the scope of the hydrolysis reaction, they are widely used in synthesis and separation chiral compounds, chemical synthesis intermediates selective protection, polymer synthesis, peptide synthesis.Recently, the enzyme catalysis in organic media have been reported numerously, but a full-cells in organic media as a catalyst for biological research is rare. Because different strain sources of the enzyme lipase have different nature and mechanisms. Study the strain of the sensitivity of different solvents lay the foundation for research in organic solvents in the catalytic reaction. On the various aspects of the research, purification of the enzyme is required.In this paper, the yield lipase strains were screened and identificated; lipase fermentation conditions were optimized; lipase was purificated and researched its basic properties. The main findings are as follows: 1. A strain of bacterium with high lipase activity was isolated form soil sample. It was identified as Serratia marcescens by physiological and biochemical detection and 16S rDNA sequence analyse, named as L-42.The strain was to tolerant to organic solvents and was can able to grow on liquid medium with 10%(v/v) acetone ,petroleum benzene,hexamethylene,glycerol. There are three isozyme EST produced by L-42.2. Factors affecting the Serratia sp.SL-11 lipase production were investigated in shake flask level. Obtained results showed that the optimum medium composition and cultural condition were(%):yeast 1.0%,soluble starch 1.0%, olive oil 1.0%, K2HPO4 0.2%, KH2PO4 0.1%; MgSO4·7H2O 0.1%, Tween 80 0.1%, and pH was 7.0,30℃, 200 r/min shaking for 48h. Under such conditions the lipase activity in culture supernatant was 2 times high as the original one.3. The lipase was purified of acetone precipitation, ion-exchange chromatography on DEAE-Sepharose and Sephadex G-100 gel-chromatography. Obtained one lipase sample, and its specific activity was 1166.61 U/mg, purified 45.57-fold, yield was 20.0%.4. Some characters of the enzyme were also in identified. The optimum temperature of lipase is 35℃and the optimum pH is 9.0.The molecular weight of the purified L-42 lipase is about 50kDa.The lipase was keenness to metal-ion , the enzyme activity was stimulated by Ca²⁺,Mg²⁺,K⁺,Mn²⁺,Ni⁺,but inhibited by Sn²⁺,Fe²⁺,Zn²⁺,Co²⁺.It indicated that lipase from L-42 would be applied widely as it showed high thermostabilities and stabilities of wide pH range.