毕赤酵母高密度发酵产Ⅲ型类人胶原蛋白及其胃粘膜修复功能

摘要

为提高重组毕赤酵母Pichia pastoris发酵重组人Ⅲ型胶原蛋白产量,采用响应面对其生长阶段的BMGY培养基(Buffered minimal glycerol-complex medium)组成进行优化.通过Placket-Burman试验,得出酵母提取物、蛋白胨、甘油对胶原蛋白产量有显著影响,通过Box-Behnken中心组合实验以及Design-Expert分析软件建立了以胶原蛋白产量为响应值的响应方程,得到最适浓度分别为酵母提取物1.13％,蛋白胨1.61％,甘油0.86％.经实验验证,在最优BMGY培养基条件下培养12 h后,所得到毕赤酵母干重为4.41 g/L,生长阶段的菌重量增加了26％.在22 L发酵罐中通过高密度发酵,产量达到4.71 g/L.该重组胶原蛋白对大鼠乙酸灼伤胃粘膜有明显修复作用,为生物医用材料的应用制备奠定了基础.%To improve collagen production by recombinant Pichia pastoris,we applied Placket-Burman and Box-Behnken design to optimize the fermentation medium.Through Placket-Burman design,three variables in the medium (concentration of yeast extract,peptone and glycerol) were selected for having significant effect on cell dry weight.Through Box-Behnken design regression coefficients analysis,a secondary degree polynomial equation was established,and the optimum levels of the three variables were yeast extract 1.13％,peptone 1.61％ and glycerol 0.86％.During the growth period,an average cell dry weight of 4.41 g/L was obtained after 12 h fermentation,increased by 26％.Through high density fermentation,the production of recombinant human collagen (Ⅲ) was up to 4.71 g/L in 22 L fermentor.The recombinant human collagen (Ⅲ) exhibited good results to repair acetic acid induced gastric ulcer in rats.