毕赤酵母表达重组葡萄糖氧化酶的发酵条件

Studies on Fermentation Conditions for r-GOD Production by Pichia pastoris GS115

对甲醇诱导重组Pichia pastorisGS115 mut+高效表达黑曲霉葡萄糖氧化酶基因的条件进行了研究,拟建立一条高效低成本的生产重组葡萄糖氧化酶的工艺路线。通过摇瓶试验对发酵培养基、诱导时机、pH、诱导温度、甲醇诱导浓度等进行了优化,随后进行了50 L发酵罐扩大化培养。结果表明,最适发酵培养基为YEPD培养基,甲醇诱导产酶的最佳时机为菌体对数生长末期,最佳诱导浓度为1%,最适诱导pH范围为6-6.5,最适诱导温度25℃。按优化的条件用50 L发酵罐分批培养酵母14 h,甲醇诱导培养48 h后生物量和酶活性达到最高。菌体终密度达到OD600为44,每升发酵液中产酶105 kU,发酵液上清蛋白含量为1 000 mg/L。

Using methanol to induce the expression of Aspergillus nigerglucose oxidase gene cloned inPichia pastorisGS115 mut＋ was deeply investigated for establishing a high-performance but low-cost method of recombinant glucose oxidase production.The medium,the pH,the time point of induction,the temperature and the methanol concentration for induction were optimized and studied in detail by shake flask experiment.The results of optimization were verified by enlarged fermentation in 50 L fermentor.Results suggested that the best medium culturing thePichia pastoriswere YEPD medium,the optimum time for induction was the mid-anaphase of logarithmic phase,the optimum inducing concentration of methanol was 1%,and the optimum pH and temperature conditions were pH6-6.5 and 25℃.According to the optimum conditions,the highest biomass and glucose oxidase expression level were obtained in 50 L fermentor with batch culture for 14 h and induced by 1% methanol for 48 h.The final cell density（OD600）was 44,105 kU enzyme activity and 1 000 mg protein were obtained in 1 L fermentation broth.