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

Production of gastric-mucosa protective collagen Ⅲ by Pichia pastoris

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。

Abstract:

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.