【摘要】 为实现环糊精酶在毕赤酵母中的高效表达,以优化合成的环糊精酶基CGT2为基础,构建组成型表达质粒pGAPZαA-CGT2,运用电转化方法将目的基因整合进酵母染色体,构建环糊精酶酵母工程菌X33/pGAPZαA-CGT2,经摇瓶发酵120h后,CGT2活力达0.21 U·mL^-1;进一步对工程菌进行摇瓶发酵条件优化,确定其最优发酵条件为:pH6.5、28℃、200r·min^-1、每24h补加2.5%的甘油,120h后其胞外酶活力达到0.36U·mL^-1,是优化前的1.7倍,实现了环糊精酶在毕赤酵母中的组成型表达。更多还原

【Abstract】 This study aimed to obtain a highly efficient expression of cyclodextrin glycosyltransferase（CGTase）in Pichia pastoris.The optimized CGT2 was cloned into a yeast constitutive expression vector,pGATZαA.The recombinant plasmid pGAPZαA-CGT2 was then transformed into P.pastoris by electroporation to construct an engineered strain,X33/pGAPZαA-CGT2.After cultivating for 120 hin a shaking flask,CGT2 with an activity of0.21U·mL^-1 was obtained.Experiments were conducted to further optimize the fermentation conditions.As a result,the greatest activity of 1.26U·mL^-1,achieving a 1.7-fold improvement,for the enzyme was reached by induction for 120 hat pH 6.5and 28℃ with a constant shaking at 200r· min^-1 and replenishing with 2.5%glycerol every 24 hin the flask.更多还原