【作者】 肖海群；

【导师】 段学辉；

【作者基本信息】 南昌大学 ， 食品科学， 2007， 硕士

【摘要】 生物柴油是一种环保型可再生的新型洁净能源，是以动、植物油脂，餐饮废油等作原料，经过与醇类（甲醇、乙醇）转酯化反应而获得的液体燃料，其具有优良的燃烧性能和环保特性、较好的发动机低温启动性能与润滑性能，可与石化柴油以任意比例混合使用。发展生物柴油对推进能源替代，控制城市大气污染，保护生态环境具有重要意义。加快生物柴油的产业化进程，对我国能源经济的可持续发展，具有现实意义。脂肪酶（Lipsae）是一类酯键水解酶，主要用于分解各种天然的油脂。脂肪酶在微生物中分布广泛，根据统计有65个属的微生物产脂肪酶。本文主要进行以下方面的研究：（1）产脂肪酶菌株的分离筛选：从南昌地区不同地点采集含油的土样50份，在以橄榄油为底物、溴甲酚紫为指示剂的平板培养基上分离、筛选到两株脂肪酶产生菌，分别命名为R32和R36。通过对菌株的形态特征、培养特征及主要生理生化特征的初步研究，确定R32为裂殖酵母属，R36为假丝酵母属，分别命名为Schizosaccharomyces R32和Candida Valida R36，摇瓶发酵的初始酶活分别为13.50U／mL、13.83U／mL。（2）菌株诱变：以Candida Valida R36为出发菌株，经过第一阶段的三轮紫外线（UV）90s剂量诱变、第二阶段的亚硝基胍500ug／mL剂量诱变、第三阶段的亚硝基胍与紫外线复合诱变后，正变率为43.5％。筛选得到一株产酶活力相对较高的突变菌株Candida Valida R36T2，摇瓶发酵酶活达到94.2U／mL。（3）发酵条件研究：通过单因素比较实验和四因素三水平的正交试验，对Candida valida R36T2进行了发酵培养基成分和发酵培养条件的优化研究。结果表明其最优摇瓶发酵培养基为（g／L）：橄榄油15.0，黄豆粉30.0，糊精10.0，NH₄NO₃10.0，MgSO₄·7H₂O 1.0，K₂HPO₄ 2.0，Tween 80 0.5；在发酵条件为：30℃，初始pH6.0，摇床转速160r／min，培养48h，细胞生物量和发酵产酶活力较高，发酵液酶活达到1467U／mL，相对出发菌株提高了近15倍。其所产脂肪酶催化油脂水解反应的最佳条件为40℃、pH7.5，Ca²⁺、Mg²⁺离子对脂肪酶的活性有一定的促进作用。更多还原

【Abstract】 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 was43.5％.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.更多还原