黏玉米谷氨酰胺酶的酵母表达及其对乳蛋白的作用

Yeast Expression of Glutaminase from Maize and Its Effects on Milk Protein

为得到大量的具有生物学功能特性的黏玉米谷氨酰胺酶,首先通过分子生物学技术将前期构建的重组质粒pPIC9K-TGase经双酶切及PCR鉴定正确后,Sac I线性化处理,电转入巴斯德毕赤酵母GS115,用体积分数为1%的甲醇诱导其分泌表达。结果:通过SDS-PAGE电泳分析,与对照组相比,在分子质量约为60 ku处,出现1条特异的蛋白带,即目的蛋白。将酵母表达TGase与0.2%酪蛋白溶液反应2 h,SDS-PAGE电泳显示,酪蛋白被聚合生成不同分子质量的聚合物,表明黏玉米TGase对酪蛋白具有很好的交联作用。将酵母表达的TGase添加到酸奶中,通过扫描电镜观察发现,添加酵母表达的TGase酸奶网络结构变得比较致密。结论:本研究获得的酵母表达的黏玉米谷氨酰胺酶具有交联乳蛋白的生物学活性。

In order to obtain a large amount of corn glutaminase with biological functions,the recombinant plasmid p PIC9K-TGase was first identified by double enzyme digestion and PCR using molecular biology techniques,then linearized by Sac I,electrotransferred into Pichia pastoris GS115,and its expression was induced by 1%methanol.Results by SDS-PAGE analysis,compared with the control,there was a specific protein band at the molecular weight of about60 ku,which was the target protein.Then,the yeast expressed TGase was reacted with 0.2%casein solution for 2hours,and SDS-PAGE showed that casein was polymerized into polymers with different molecular weights,indicating that the expressed protein had a good cross-linking effect on casein.Moreover,the TGase from yeast expressed was added to yogurt,and it was observed by scanning electron microscope that the yogurt network structure with yeast expressed TGase became relatively dense.Conclusion:The yeast expressed glutaminase has the biological activity of crosslinking milk protein.