【作者】 邱慧；

【导师】 唐云明；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 脂肪酶（EC3.1.1.3,甘油酯水解酶）是一类能催化长链脂肪酸甘油酯水解为甘油和长链脂肪酸（或者是此反应的逆反应）的生物催化剂.它可以在油水界面上催化甘油三酯水解生成脂肪酸和甘油,以及中间产物甘油一酯和甘油二酯.广泛应用于食品、医药、洗涤、皮革、造纸等行业领域.本文以实验室保存的碱性脂肪酶高产诱变菌株Lp-11为出发菌株,通过菌种复壮.产酶条件优化,从而提高菌株产酶量.并对其碱性脂肪酶进行高效分离纯化,以期降低纯化成本.提高碱性脂肪酶的活性回收率.利用平板划线法和平板涂布法对菌株Lp-11进行菌种复壮.得到一株产脂肪酶活性相对最高且传代稳定的菌株:Lp-11-④。利用逐因子试验及正交设计等方法对菌株Lp-11-④进行产酶条件的优化。首先运用逐因子试验确定Lp-11-④产碱性脂肪酶的最适碳源和氮源分别为:葡萄糖和蛋白胨.然后在此基础上利用正交试验对其发酵培养基成分进行优化,最后得到其最佳培养条件为:葡萄糖0.5%;蛋白胨5.0%;Na₂HPO₄0.7%;KH₂PO₄0.5%;Tween80 1.0%:橄榄油2.0%;培养基pH8.0:接种量3.0%（v/v）:种龄12h:转速250r/min;温度30℃,在此条件下培养（30h）发酵液酶活达到366.996U/ml,比优化前提高84.541%.对菌株Lp-11-④所产的碱性脂肪酶进行高效分离纯化.菌体发酵液经双水相萃取（ATPS）、Phenyl-Sepharose疏水作用层析处理.超滤浓缩后得到了纯化后的碱性脂肪酶,用SDS聚丙烯酰胺凝胶电泳和等电聚焦检测其纯度为电泳纯.该酶的纯化倍数为26.81倍.活性的回收率为65.15%.比活力达到9901.02U/mg.理化性质研究表明,脂肪酶的分子量为265.6KD,由四个分子量为64.8KD的相同亚基组成,该酶的等电点pI为5.43。更多还原

【Abstract】 Lipase（EC3.1.1.3,triacylglycerol acylhydrolase）is a kind of biocatalysts to hydrolysis long-chain triglycerides to glycerol and lang-chain fatty acids.It can hydrolysis Triglyceride to fatty acid and glycerol in the interface of oil-water.Lipase is widely used in food,medicine,scour,leather and paper industry.In this research work,rejuvenation and enzyme production optimization were used to improve the lipase production of Lp-11-④.Alkalin lipase was purified high efficiently to reduce purification costs.We used plate streaking and coating methods to rejuvenate Lp-11 which was conserved by our laboratory.Finally,the high yield Lp-11-④strain was selected.Stability test demonstrated that the lipase activity remained the same level after five generations.Lipase production condition of Lp-11-④was optimized using seriatim-factorial experiment and orthogonal experiment.The optimum carbon source and the optimum nitrogen source for Lp-11-④screened through seriatim-factorial experiment were glucose and peptone,respectively.By orthogonai experiment we obtained result of the optimum culture medium was:glucose0.5%; peptone5.0%;Na₂HPO₄0.7%;KH₂PO₄0.5%;Tween80 1.0%;olive oil2.0%;pH8.0;inoculation quantity 3.0%（V/V）;bacteria age 12h;revolution 250r/min;temperature 30℃.After 30h incubation with the optimum condition,a maximum lipase yield 366.996U/mL was obtained,which was 84.541%higher than that under beginning culture mediums.The alkaline lipase of the supernatant was purified to homogeneity through aqueous two-phase extraction and hydrophobic interaction chromatography on Phenyl-Sepharose Fast Flow column, and the pure alkaline lipase was characterized by SDS-PAGE and IEF electrophoresis.65.15%of the lipase activity was recovered,and purification fold was 26.81.The specific activity was 9901.02 unit/mg.The molecular weight of the lipase is 265.6KD and submits are 64.8KD determined by gel filtration chromatography on Superdex-200 and SDS-PAGE method.Isoelectric point of the pure lipase was 5.43 showed by IEF-PAGE.更多还原