果胶寡糖生产菌的构建与筛选

Construction and screening of recombinant yeast producing pectin oligosaccharides

生物酶解法是新兴益生元果胶寡糖(Pectin oligosaccharides,POS)生产的主要途径.通过构建黑曲霉来源的多聚半乳糖醛酸酶(Polygalacturonase,PGA)基因pga A表达载体p PIC9K-PGA,电转毕赤酵母GS115,筛选能够表达用于制备POS重组果胶酶的工程菌.利用SDS-PAGE检测诱导表达上清,TLC分析其水解产物成分,筛选得到一株特定降解果胶生成寡糖的酵母工程菌GS-12.该重组多聚半乳糖醛酸酶(Recombinant polygalacturonase,re PGA)相对分子量(Mr)为86×10~3,发酵液酶活水平24.8 U/m L;GS-12经30次传代后目的基因pga A仍能稳定遗传,水解产物成分未发生改变;酶促反应得出该酶最适温度50℃,最适p H 5.5,50℃保温5 h相对酶活保持50%以上.综上,所构建酵母工程菌GS-12遗传稳定性高,耐热性提升,作用果胶可得大分子量多糖产物,对POS的工业规模化生产具有重要价值.

Hydrolysis conducted using recombinant pectinase is one of the best ways to produce pectin oligosaccharides （POS）. In this study, a secretory expression plasmid pPIC9K-PGA carrying polygalacturonase gene from Aspergillus niger （pgaA） was successfully constructed and transformed into Pichia pastoris GS115 to obtain the recombinant strain named GS-12. In addition, the hydrolysis product of recombinant polygalacturonase （rePGA） expressed by GS-12 was analyzed using thin layer chromatography. After the expression of the target gene was induced in GS-12, the rePGA was detected using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was 86 × 10^3 and crude enzyme activity was 24.8 U/mL. The characterization of rePGA showed that its optimal reaction condition was 50 ℃ at pH 5.5. After incubation at 50 ℃ for 5 h, above 50% of the relative activity was retained. Further, the expression of pgaA was stable in GS-12 after 30 generations. Thus, in this study, a recombinant yeast GS-12 with good inheritance stability was obtained. The molecular weight and thermal stability of rePGA were higher than the predicted value, but its hydrolysis activity decreased, and high-molecular-weight saccharides were obtained after the degradation of pectin. The results of this study provide valuable indicative information for the production of POS.