Mutagenesis And Strain-improving Of Alkaline Lipase Producing Strain, Purification And Immobilization Of The Alkaline Lipase

Lipase is widely used in the hydrolyzation, alcoholysis and synthesis of ester, polypeptides, macromolecular polymer and the splitting reaction of enantiomorph polymer in the interface of oil-water. It is one kind of most applied enzymes in food, medicine, scour and leather.In this research work, various ways of mutagenesis treatment, such as UV-irradiation, MW, NTG and DES, were carried to study the effectiveness of increasing the productivity of lipase producing strain Serratia sp.SL-11 which was conserved by our laboratory. Finally, the high yield mutant (obtained from DES treatment) was selected and produced alkaline lipase activity 75% higher than that of parent strain. The fermentation potency of the mutant was 198U/mL in shaker. Stability test demonstrated that the lipase activity remained the same level after five generations.The alkaline lipase of the supernatant was purified to homogeneity through hydrophobic interaction chromatography on Phenyl-Sepharose Fast Flow column and ion exchange chromatography on DEAE-Sepharose Fast Flow column, and the pure alkaline lipase was characterized by SDS-PAGE and IEF electrophoresis. 39.61% of the lipase activity was recovered, and purification fold was 31.96. The specific activity was 5752.12unit/mg. The molecular weight of the lipase is 292.3KD and submits are 70.9KD determined by gel filtration chromatography on Superdex-200 and SDS-PAGE method. Isoelectric point of the pure lipase was 5.48 showed by IEF-PAGE.The alkaline lipase were selectively modified by pCMB, pMSF, NBS, NAI, BrAc, DTT , BD, maleic anhydride and chloramine-t. It was found that the reaction of lipase with NBS, maleic anhydride and chloramine-t resulted in a strong inhibition of enzyme activities which decreased with the increase of modifier concentration. pCMB, pMSF, NAI, BrAc, DTT and BD had no effect on the activity of alkaline lipase. Studies of inactivation of lipase by chemical modification demonstrated that Trp, Lys and Met residues are essential functional groups of the alkaline lipase.There are many limitations in application of free enzyme. So in this paper, immobilization of alkaline lipase was studied. The immobilization conditions such as supporting materials, pH value, temperature, the amount of lipase added, the concentration of sodium alginate, the concentration of calcium chloride and the time of immobilization were determined. The result showed that the maximum immobilized lipase activity of 280unit/g was obtained at the following conditions: the ratio of Phenyl-Sepharose to lipase being 10:1(g/mg), pH value of Tris-HCl buffer solution being 8.0, the best temperature being 50℃, the time of immobilization being 1 hour.