The optimal conditions for producing chymosin in 5L fermenter by recombinant Pichia pastoris Gsl 15/pPICZaA-Prochy were determined in order to offer technical bases for pilot scale production. After induction for 84h, the highest enzyme activity (152SU/ml) was achieved with batch culture, in which, lOg/L sorbitol was added at the beginning of induction phase, methanol concentration was kept at 0.1%, 10g/l peptone was added every 12h, pH value was kept at 3.0 and the concentration of oleic acid was 0.2%. The purification of chymosin was analyzed by sodium dodecyl sul-fete benzene agarose gel electrophoresis (SDS-PAGE). Under the concentration of salt ions at 2mol/L and flow rate 2.5mL/min, the recovery rate of chymosin was 82.33% and the enzyme sample was concentrated by 20 folds.%以重组毕赤酵母GS115/pPICZaA-Prochy为凝乳酶生产菌种,研究其在5L发酵罐水平的最佳产酶条件,为中试生产提供依据.采用批量发酵,甲醇浓度0.1％,诱导阶段每12h添加10g/L蛋白胨,发酵液pH值为3.0,油酸浓度0.2％,诱导初期批量加入10g/L山梨醇共基培养,诱导84h可获得152SU/mL的酶活力.采用十二烷基磺酸钠-苯琼脂糖凝胶电泳分析,在发酵液(去菌体后)样品盐离子浓度2mol/L,流速2.5mL/min,酶回收率达到82％,浓缩倍数20倍.
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