Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris
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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120 h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℃、200 r·min-1、每24 h补加2.5%的甘油，120 h后其胞外酶活力达到0.36 U·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。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 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℃ with a constant shaking at 200 r·min-1 and replenishing with 2.5% glycerol every 24 h in the flask.
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图 1 重组表达载体pGAPZαA-CGT2菌落PCR验证
注：M为DL 1 kb DNA Marker；1~2为Colony PCR of CGT2。
Figure 1. PCR validation on pGAPZαA-CGT2colony
图 2 重组酵母基因组PCR鉴定结果
注：M为DL1kb DNA Marker；1~6为PCR of CGT2。
Figure 2. PCR of recombinant P. pastoris
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