【作者】 张大皓；

【导师】 谭天伟；

【作者基本信息】 北京化工大学 ， 生物化工， 2006， 硕士

【摘要】 卡门柏青霉—PG3脂肪酶是一种具有1,3位置专一性的微生物脂肪酶。本文对该脂肪酶的生产工艺进行了研究。用相应面法优化了霍霍巴油作为碳源时的培养基,但是霍霍巴油价格太高,不利于工艺放大。所以重点优化碳源,最后得出结论,不加入专门碳源的情况下产酶水平也较高。从降低发酵成本和分离提取工艺的简化两方面考虑,采用不加入专门碳源的培养基作为基础培养基,对此培养基优化得到(g/L)脱脂豆粕40,(NH4)2HPO41, K2HPO45,吐温601,温度28℃,转速220rpm,自然pH在250mL摇瓶规模的发酵水平达到230IU/mL左右。在250mL中进行补料实验,在48和72h分别补加1g脱脂豆粕使最高酶活达到380IU/mL。在5L发酵罐中进行高浓度匀速流加实验,发酵条件(g/L)：脱脂豆粕40,(NH4)2HPO41, K2HPO45,吐温601,消泡剂1,硫酸庆大霉素4万单位/L,温度28℃,转速220rpm, pH自然,在48—115h内补加1L含120g/L豆粕的补料液最终酶活达到392IU/mL。对卡门柏青霉PG3脂肪酶的分离提取工艺进行研究,直接采用丙酮沉淀的方法,最佳丙酮用量为3倍体积,收率达到74.8%,相对酶活达到8681IU/mL。对粗酶的基本性质进行研究,最适酶反应温度43℃,最适pH7.0。在pH中性到碱性范围内酶稳定性较好。更多还原

【Abstract】 The production of Penicillium camembert ii Thom PG-3lipase, which is known for the1,3-regioselectivity on the hydrolysis of triglyciates, were studied, and the techniques for the lipase production were improved. The fermentation medium constitution in which jojoba (one species of natural oil) served as carbon source was optimized using Response Surface Methodology (RSM). However, it was found that the culture were not suitable for large-scale production because of the expensive jojoba. Further research indicated that the lipase production could be considerably satisfying without the involvement of carbon source. Considering both cost and efficiency of the ferment process, the medium composition was optimized without manually introduced carbon source, and the optimum culture conditions were confirmed as (g/L):defatted soybean meal40,(NH4)2HPO41, K2HPO45, tween-601, cultured at28℃,220rpm;230IU/mL enzyme activity was gained in flasks without pH adjustment, and higher enzyme activity (380IU/mL) was observed when1g defatted soybean meal was fed at48h and72h respectively. Scale-up fermentation was carried out in a5L fermentor, and high concentration of the soybean meal was fed at constant rate of120g/L in48-115h. Final enzyme activity reached392IU/mL using this strategy. The culture in the fermentor was conducted under following conditions (g/L):defatted soybean meal40,(NH4)2HPO41, K2HPO45, tween-601, antifoam1, gentamicin sulphate40000u/L, ambient pH.Separation of Penicillium camembert ii Thom PG-3lipase was attempted using precipitation by organic solvents. Herein3equivalents acetone of the broth volume was most favorable for precipitation with crude yield of74.8%and enzyme activity8681IU/mL. Initial studies in the enzyme properties showed that optimum conditions for enzymatic transformation were43℃at pH7.0; the lipase remained stable within pH range from neutral to basic.更多还原