重组α-1,3-葡萄糖苷酶的毕赤酵母表达及发酵优化

Expression in picha pastoris and optimization of fermentation of recombinant α-1,3-glucosidase

Acremonium sp.S4G13来源的α-葡萄糖苷酶基因连接到p PIC9K载体上并在Pichia pastoris GS115中表达。在此基础上对重组菌P.pastoris GS115/pPIC9K-AGL产重组α-葡萄糖苷酶的发酵条件进行了单因素优化试验,结果表明最优条件为:生长阶段接种量10%,甘油体积浓度3%,初始pH 6.0;诱导阶段甲醇初始添加量1%,甲醇补加量0.5%,装液量10%,诱导温度25°C,山梨醇添加量6 g/L。在最优条件下发酵120 h,酶活可达7.8 U/mL,相比优化前酶活1.22 U/mL提升了5.4倍。

The gene encodingα-glucosidase from Acremonium sp.S4 G13,agl,was introduced into the p PIC9 K plasmid,and then transformed into Pichia pastoris GS115 to achieve the secretive extracellular overproduction ofα-glucosidase.Single-factor optimization experiments were performed on the fermentation conditions related to the recombinantα-glucosidase by P.pastoris GS115/pPIC9 K-agl.The optimum fermentation conditions were determined as follows:incolumn size 10%(v:v),concentration of glycerin 3%(v:v),initial pH 6.0,initial methanol concentration 1%(v:v),methanol supplementation quantity 0.5%(v:v),inducing temperature 25°C,the bottled fluid volume 20%(v/v),concentration of sorbitol 8 g/L.Optimized enzyme activity was reached 7.8 U/mL at the optimum conditions after fermentation for 120 h,which was 5.4 fold higher than the initial 1.22 U/mL.