毕赤酵母表面展示磷脂酶D高密度发酵优化

High Density Fermentation Optimization of Phospholipase D Displayed on Pichia pastoris Cell Surface

利用基因工程手段构建获得的一株细胞表面展示表达磷脂酶D的毕赤酵母工程菌株GS115/pKFS-pld,通过摇瓶单因素筛选进行发酵条件优化,确定了最佳发酵条件为:诱导产酶阶段28℃,初始pH6.5,甲醇浓度1.5%,装液量为30 mL/250 mL,250 r/min摇床培养144 h。在此优化条件下菌体量为19.6 g/L,酶活达17.8×10-7kat/g,分别是是优化前的1.38及1.44倍。同时进行了5 L规模发酵罐分批补料培养,结果表明15 mL(/L.h)速率流加甘油补料培养基6 h后,采用溶氧恒定流加法流加甲醇,诱导132 h后,菌体量及PLD活力分别达到67.4 g/L及27.3×10-7kat/g,是摇瓶发酵水平的3.44倍及1.53倍。

The BMGY/BMMY medium and fermentation conditions of phospholipase D（PLD） displaying engineered strain Pichia pastoris GS115/pKFS-pld were optimized in single factor shake flask levels. The results indicated that the optimum fermentation conditions of GS115/pKFS-pld were the combination of the induced temperature 28 ℃, induced initial pH 6.5, methanol 1.5 %（v/v）, medium volume 30 mL/250 mL, and shaking at 250 r/min. Under these conditions, the PLD activity and DCW of GS115/pKFS-pld were significantly higher to 17.8x10-7 kat/g and 19.6 g/L, which was increased by 38 % and 44 %, respectively, compared to the original conditions. Meanwhile, the optimization of culture conditions on GS115/pKFS-pld was scale-up in the 5 L fermentor. The biomass was rapidly enriched effectively when the glycerol fed-batch phase flow rate was setted at 15 mL/（L·h） and the flow time of glycerol was extended to 6 h, which could effectively increase the latter induced expression of PLD. By the methanol fed-batch stage using DO-star method, the PLD activity and DCW of GS115/pKFS-pld reached 27.3 ×10^-7 kat/g and 67.4 g/L, which was 1.53 and 3.44 times of that the results in the shake, respectively.