环糊精酶基因在毕赤酵母中的组成型表达  

Constitutive Expression of Cyclodextrin Glycosyltransferase in Pichia pastoris

作　　者：陈龙军[1] 陈济琛[1] 林新坚[1] 蔡海松[1] 

机构地区：[1]福建省农业科学院土壤肥料研究所,福建福州350003

出　　处：《福建农业学报》2017年第1期82-86,共5页Fujian Journal of Agricultural Sciences

基　　金：福建省科技计划项目--省属公益类科研院所基本科研专项(2014R1022-3;2015R1022-1);福建财政社会公益研究(2060302);国家公益性农业科研专项(201303094-05)

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。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.

关 键 词：环糊精酶 毕赤酵母 组成型表达 发酵 

分 类 号：Q786[生物学—分子生物学]