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

作　　者：刘逸寒[1] 薄嘉鑫[1] 乔婧 张超 王建玲[1] 路福平[1] 

机构地区：[1]工业发酵微生物教育部重点实验室工业酶国家工程实验室天津市工业微生物重点实验室天津科技大学生物工程学院,天津300457

出　　处：《食品研究与开发》2012年第8期184-187,共4页Food Research and Development

基　　金：十二五"农村领域国家科技计划(2011AA100905-4);国家自然科学基金(31101219)

摘　　要：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.

关 键 词：毕赤酵母表面展示 磷脂酶D 磷脂酰丝氨酸 发酵优化 酶活力 

分 类 号：TQ925[轻工技术与工程—发酵工程]