基于模拟酶切的大豆7S球蛋白酶解改性探究

Modifications of soy 7S globulin using enzymatic hydrolysis based on in silico analysis

为高效筛选适用于改善大豆7S球蛋白酸溶特性的蛋白酶,该文利用PeptideCutter软件对7S进行模拟酶切,然后采用Compute pI/M_(w)软件预测生成肽段的等电点(isoelectric point,pI),从中挑选pI>pH 6.0或pI<pH 3.0的肽段建立肽库,结果发现在39种选定蛋白酶中能得到20个以上目标肽段的蛋白酶依次分别是蛋白酶K、胃蛋白酶、嗜热菌蛋白酶、胰凝乳蛋白酶和胰蛋白酶。实验验证表明,上述5种优势酶都可不同程度提高7S在pH 3.0~4.5下的蛋白溶解度(protein solubility,PS),其中蛋白酶K表现最佳,其制备的7S酶解产物的PS在pH 3.0~5.0下约为81%。进行电泳和Zeta电位分析发现与模拟酶切预测结果一致,5种优势酶可将7S中α′、α和β亚基完全降解成小分子肽,所得7S酶解产物等电点与对照7S相比向酸性方向偏移。但由于7S酶解过程的复杂性,当不同蛋白酶模拟酶切所得目标肽段数量相差不大时,预测结果不够准确。采用Turbiscan稳定性分析仪测定发现7S经蛋白酶K酶解改性后在酸橙汁(pH 3.86)中不易引起絮凝沉淀。该研究表明基于计算机模拟酶切指导7S酶解改性可大大提高水解用酶的筛选效率,所制备的7S酶解产物具有良好的酸溶特性,在酸性饮料中有应用前景。

To efficiently select proteases appropriate for improving the protein solubility of soy 7S globulin under acidic conditions,7S was proteolyzed in silico using the software PeptideCutter,and then the isoelectric points(pI) of produced peptides were predicted using the on-line software Compute pI/M_w.Peptides with pI>pH 6.0 or pI<pH 3.0 were selected to construct peptide library,whereby the appropriate proteases were screened and were verified through in vitro experiments.Results showed that proteases that could produce 20 or more target peptides from in silico analysis were protease K,pepsin,thermolysin,chymotrypsin,and trypsin in sequence.Results of in vitro experiments verified that these five proteases could improve the protein solubility(PS) of 7S at pH 3.0-4.5 to varying degrees,with protease K performing the best.The PS of 7S hydrolysates modified by protease K was approximately 81% at pH 3.0-5.0.Analysis of electrophoresis and Zeta potential of 7S hydrolysates demonstrated that the α′,α and β subunits of 7S underwent total degradation after proteolysis using the five advantageous proteases,and the resultant 7S hydrolysates mainly consisted of peptides with molecular weights<3 000 Da,and showed a shift in pI to acidic conditions.These findings were consistent with those of in silico analysis.However,due to the complexity of proteolysis of 7S,the in silico analysis was insufficiently accurate when the quantities of target peptides produced by different proteases were not much different.Stability analyzer results showed that 7S hydrolysate modified by protease K was more stable against flocculation and precipitation than the control 7S in orange juice.This study demonstrated that based on in silico analysis,the screening efficiency of appropriate proteases for enzymatic modifications of 7S could be increased markedly,and the resultant 7S hydrolysates showed good protein solubility under acidic conditions and have a potential application prospect in acidic beverages.