富含多酚的麻栎种子中蛋白质提取的探讨

Protein Extraction from Polyphenol-Rich Seeds of Quercus acutissima

采用单因素试验和正交试验的方法,对影响麻栎种子中蛋白质提取的主要因素进行分析,找出最佳提取条件,即采用含有1mmol.L-1EDTA,1mmol.L-1AsA,1mmol.L-1DTT,1mmol.L-1GSH,5mmol.L-1MgCl2,0.05%TritonX_100和20%甘油的50mmol.L-1磷酸钾缓冲液(pH值7.5),加入与材料等质量的不溶性聚乙烯多聚吡咯烷酮(PVPP)与材料共同研磨,即可获得大量的蛋白质。其中最主要的影响因素是PVPP的用量,PVPP阻止了多酚与蛋白质的结合,并将多酚沉淀,从而增加了蛋白质的溶解性。

The seeds of Quercus acutissima are rich in polypbenols, which cause protein precipitation and coloration of extracts. Because of this precipitation, proteins in seeds of Q. acutissima cannot be extracted using common methods. Here we report an optimized method for the seed protein extraction. After being surface dried for 12 h at room temperature, seeds were sliced on ice and ground to powder in a mortar under liquid nitrogen, and then kept at - 70 ℃ before use. The extraction method was studied by mono-factor experiments and orthogonal experimental design. The effects of different kinds of extraction buffers, their volumes and pH values and the amounts of PVPP （polyvinylpolypyrrolidone） were studied. The results of monofactor experiments indicated that all these factors affect the yield of protein extracted from the seeds. Protein was extracted with 2, 5 and 10 volumes of extraction buffer respectively, and we obtained more protein when using 5 volumes of extraction buffer compared to 2 or 10 volumes. Phosphate buffers were superior to Tris-HCl buffers, and higher pH values and PVPP amounts were better than lower ones. The results of orthogonal experiments showed that the amount of PVPP had the greatest effect on protein extraction among all the factors discussed in the paper. The optimal method for protein extraction from seeds of Q. acutissima was potassium phosphate buffer （ 50 mmol·L^-1, pH7.5）, containing 1 mmol ·L^-1 EDTA, 1 mmol·L^-1 AsA, 1 mmol·L^-1 DTT, 1 mmol·L^-1 GSH, 5 mmol·L^-1 MgCl2, 0.05%Triton X-100 and 20% glycerol, and equal amounts of PVPP to fresh weight of the material. And furthermore the material should be ground together with PVPP, thus PVPP can adsorb and precipitate polyphenol preventing it from binding with protein as early as possible, and PVPP can also help homogenize the material thoroughly.