不同pH值对木瓜蛋白酶活性的影响及分子机制  

Effect of Different pH on Papain Activity and Its Molecular Mechanism

作　　者：代雯 黄业传 韩佳钰 胡霄 李典典 管思彤 DAI Wen;HUANG Yechuan;HAN Jiayu;HU Xiao;LI Diandian;GUAN Sitong(College of Food and Biology,Jingchu University of Technology,Jingmen 448000,China)

机构地区：[1]荆楚理工学院食品与生物学院,湖北荆门448000

出　　处：《肉类研究》2024年第10期9-14,共6页Meat Research

基　　金：荆楚理工学院校级创新创业项目(KC2023052);荆门市重点科技计划项目(2022YFZD058)。

摘　　要：为探究不同pH值对木瓜蛋白酶活性的影响及分子机制,采用分子模拟和荧光光谱分析等技术,对不同pH值条件下(4、5、6、7、8)木瓜蛋白酶的酶活性、均方根误差(root mean square deviation,RMSD)、蛋白质残基的波动、回旋半径、蛋白间氢键、蛋白质二级结构和溶剂可及表面积等进行分析。结果表明:pH7时木瓜蛋白酶活性最高,pH4和pH8时比pH7时活性分别降低约5.00%和5.35%;分子模拟结果显示,不同pH值下木瓜蛋白酶的回旋半径波动不大,pH8时RMSD有较大波动;pH4和pH8时蛋白酶活性中心的3个氨基酸残基有较大波动;随着pH值的增加,木瓜蛋白酶间氢键数量逐渐增加,但pH8的氢键数量较pH7下降3.4个;pH7条件下木瓜蛋白酶活性中心与底物结合紧密,酶蛋白内部结构更致密,而pH4条件下酶活性中心与底物结合部位缝隙较大,酶蛋白内部结构疏松;pH4和8时活性下降与该条件下木瓜蛋白酶的结构变化有关,如氢键的破坏、活性中心残基的波动、疏水表面积的改变、β-折叠等二级结构的变化等。本研究从分子模拟角度明确了pH值对木瓜蛋白酶活性的影响。In order to investigate the impact of different pH levels on papain activity and its molecular mechanism,molecular simulation and fluorescence spectroscopy were employed to analyze the enzyme activity,root mean square error(RMSE),root mean square fluctuation(RMSF),radius of gyration(Rg),inter-protein hydrogen bonding,protein secondary structure,and solvent accessible surface area at various pH values(4,5,6,7,and 8).The results revealed that papain activity was highest at pH 7 and decreased by approximately 5.00%at pH 4 and 5.35%at pH 8 compared to pH 7,respectively.Molecular simulation results indicated that the Rg of papain exhibited minimal fluctuations across different pH levels;RMSD fluctuated greatly at pH 8,and three amino acid residues in the active center displayed significant fluctuations at both pH 4 and pH 8.As the pH value increased gradually,the number of hydrogen bonds in papain also increased except for a decrease by 3.4 at pH 8 compared to pH 7.Under a neutral condition(pH 7),the active center of papain tightly bound to its substrate with a denser internal protein structure;whereas under acidic conditions(pH 4),there was a larger gap between protease’s active center and its substrate binding site along with a looser internal protein structure.The decline in the activity observed at low pH(pH 4)and high pH(pH 8)could be attributed to structural changes protease such as disruption of hydrogen bonds,fluctuations in active center residues,alterations in hydrophobic surface area,and modifications in secondary structures such asβ-sheet.This study clarified the effect of pH on papain activity from a molecular simulation perspective.

关 键 词：木瓜蛋白酶 酶活性 PH值 分子模拟 

分 类 号：TS201.1[轻工技术与工程—食品科学]