Studies On Breeding Of Lipase-producing Strains And Fermentation Conditions For Lipase Producing

Biodiesel is a novel energy, which is reproduceable and friendly environment.Biodiesel is prepared by transesterification of vegetable oils, animal fats or wastecooking oil with alcohols. Biodiesel has excellent physical-chemical characteristics,such as good combustion and environmental protection, low temperature cranking andlubricity. And in application it can be mixed with petrifaction diesel oil at arbitraryproportion.Development of biodiesel is important for forwarding substitutional energysource, controlling urban air pollution and protecting ecological environment. Andimproving the industrial process of biodiesel has realistic meaning for sustainabledevelopment of Chinese energy economy.Lipase is a kind of hydrolase for ester bond hydrolyzation, which can be mainlyused to break down various kinds of natural fats. And lipase exists widely in microbialorganisms. Research data showed that there are about 65 genera microorganismslipase-producing.Main contents discussed in this thesis are as follows:(1) Isolation and screening of lipase-producing strains: From 50 soil samples,two lipase-producing strains are isolated by the primal screening on agar platescontaining olive oil and bromcresol purple. And the two strains are labeled R32 andR36 respectively. After analyzing and comparating of microorganism morphology,physiology and biochemistry propterties, the strain R32 and R36 were identified asSchizosaccharomyces and Candida Valida and named Schizosaccharomyces R32 andCandida Valida R36. Their initial lipase activities reached 13.50U/mL and13.83U/mL respectively.(2) Mutation of lipase-producing strains: To improve Candida Valida R36'slipase-producing activity, it was mutated with UV, NTG and composite mutation ofUV and NTG. The experiment result showed that the positive mutation rate was43.5ï¼….Further screening was implemented with monitoring of lipase-producingactivity. A strain with comparatively high lipase yield was selected. Its enzymeactivity reached 94.2 U/mL in shaking flask fermentation and named Candida ValidaR36T2.(3) Study on fermentation conditions of lipase-producing strains: By single factor comparison experiment, four-factor and three-level orthogonal experiment, mediumcomposition and fermentation conditions of swain Candida Valida R36T2 wereoptimized. The results showed that the optimal medium composition for producinglipase by Candida Valida R36T2 is (g/L): olive oil 15.0, soybean meal 30.0, dextrin10.0, NH₄NO₃ 10.0, MgSO₄·7H₂O 1.0, K₂HPO₄ 2.0,Tween 80 0.5. In above condition,the biomass of cell and enzyme activity produced by Candida Valida R36T2 are best.The enzyme activity of fermentation aqueous solution reached 1467U/mL under thefermentation conditions with 30℃, initial pH 6.0 and shaking speed of 160r/min for48h. The lipase activity was more fifteen times than that of the original strain isolated.Besides, calcium ion and magnesium ion can play some promoting effects on lipaseactivity. And the mutant strain---Candida valida R36T2 screened has stableinheritance.