毕赤酵母高密度发酵生产白地霉脂肪酶研究生物工程专业论文.docxVIP

华中科技大学硕士学位论文 华 中 科 技 大 学 硕 士 学 位 论 文 II II g/L，酶活力达到 6,500 U/mL，酶活生产率达到 38.62 U/(mL·h)。 （3）将实验室构建的双启动子重组菌 GS115/9Kgcl-FLDgcl 26#进行 10-L 发酵罐 混合碳源补料发酵研究。以甲醇和山梨醇作为混合碳源进行诱导，发酵 113.1h，生 物量达到 396 g/L，蛋白含量达到 4.52 g/L，酶活力达到 11,200 U/mL，酶活生产率达 到 98.98 U/(mL·h)。 （4）对比三株工程菌在 10-L 发酵罐中的发酵特性及产酶情况，结论为：Muts 型参数变化较 Mut+型更稳定；甲醇与山梨醇混合补料可以明显减少色素（FAD）的 产生；采用甲醇与山梨醇混合补料相对甲醇单独补料，酶活力提高 20%，酶活生产 率提高 30%~60%；而采用分子手段构建的双启动子工程菌，其酶活相对单一启动子 工程菌提高约 1 倍，酶活生产率也提高 1 倍左右。 关键词：白地霉脂肪酶；毕赤酵母；摇瓶优化；高密度发酵；混合补料 III III Abstract The Geotrichum candidum lipase (GCL) is a catalyst with substrate specificity to the fatty acids owning a cis-double bond in the 9-position. Thus, GCL can be used to selectively hydrolyze fatty acids to enrich unsaturated fatty acids (UFAs) with high nutritional value or to selectively esterificate and separate conjugated linoleic acid isomers. The objectives of this thesis are to optimize shake flask fermentation conditions and high-density fermentation conditions of the recombined Pichia pastoris harboring GCL B gene and appearing Mut+ phenotype and Muts phenotype, for enzyme production in 10-L fermenter. The main results are as follows: Rhodamine B flat and shake flask fermentation were employed to quanlitatively and quantitatively detect the enzyme activity of X-33/pPICZα-gcl 57#. The initial enzyme activity is 140U/mL (olive oil emulsion, alkali type titration). The optimized shake flask fermentation conditions were 40 mL culture medium in 500 mL shake flask, pH 7.0, 1% methanol, and induced 72 hours with the highest enzyme activity at 375 U/mL. Then, the fermentation conditions of X-33/pPICZα-gcl 57# in 10-L fermenter were optimized, and the optimal conditions of enzyme production were: fermentation temperature 29.0℃, pH 5.0 in cell growth phase, and the temperature 27.0℃, pH 6.0 in the induction phase. Under such conditions, the biomass reached 338 g/L, the protein content 2.68 g/L, the enzyme activity 5,000 U/mL, and the productivity of enzyme activity 30.69 U/(mL?h) after 16