环糊精酶基因在毕赤酵母中的组成型表达

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。

Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris

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 h in a shaking flask,CGT2 with an activity of 0.21 U · mL-1 was obtained.Experiments were conducted to further optimize the fermentation conditions.As a result,the greatest activity of 1.26 U · mL-1,achieving a 1.7-fold improvement,for the enzyme was reached by induction for 120 h at pH 6.5 and 28℃C with a constant shaking at 200 r · min-1 and replenishing with 2.5％ glycerol every 24 h in the flask.