乳化法制备毕赤酵母微胶囊的条件优化

Optimization conditions for preparation of Komagataella phaffii microcapsule by emulsificaton

毕赤酵母(Komagataella phaffii,原名Pichia pastoris)是一种甲基型营养酵母,其表达系统是当前最重要的外源蛋白表达系统之一。重组毕赤酵母表达外源蛋白的过程研究通常采用细胞群体分析,获得大量细胞的平均化结果,但无法区分不同细胞个体的性能差异性。本研究以表达绿色荧光蛋白(GFP)的重组毕赤酵母GS115-gfp为材料,海藻酸钠为壁材,通过内源乳化法将毕赤酵母GS115-gfp包埋进海藻酸钠微胶囊中,对微胶囊内的毕赤酵母GS115-gfp进行诱导表达。结果表明在钙离子与海藻酸钠摩尔质量比为1∶3,乙酸与海藻酸钠的摩尔质量比为1∶3,Span 80添加量为1.5%,水相与油相比为0.4665,转速为500 r/min的条件下制备所得微胶囊的粒径最小(93.03μm),此时包埋率为80.01%。利用甲醇诱导所制备的毕赤酵母微胶囊,可以表达GFP。本研究为高效表达外源蛋白的重组毕赤酵母筛选奠定了基础。

Komagataella phaffii(formerly known as Pichia pastoris),is a kind of methylotrophic yeast,its expression system is one of the most important exogenous protein expression systems at present.The process of heterologous proteins expression by recombinant Komagataella phaffii was usually carried out using cell population analysis,which obtained average results of a large number of cells,but could not distinguish the variability of different individual cells.In this research,using recombinant Komagataella phaffii GS115-gfp expressing green fluorescent protein(GFP)as material and sodium alginate as wall material,Komagataella phaffii GS115-gfp was encapsulated into sodium alginate microcapsules by endogenous emulsification method,and the expression of Komagataella phaffii GS115-gfp was induced in microcapsules.The results showed that the microcapsules were prepared at the molar mass ratio of calcium ion to sodium alginate 1∶3,the molar mass ratio of acetic acid to sodium alginate 1∶3,the Span 80 addition 1.5%,the aqueous phase to oil 0.4665,and the rotational speed of 500 r/min.The resulting microcapsules had the smallest particle size(93.03μm),with the embedding rate of 80.01%.GFP could be expressed in Komagataella phaffii microcapsules prepared using methanol induction.This study laid the foundation for the screening of recombinant Komagataella phaffii with highly efficient expression of heterologous proteins.