Based on the three-dimensional (3D) structure of trypsin, 2-nitrophenyl-β-D-glucopyranoside was selected from the ZINC database to be a candidate affinity ligand for trypsin. The affinity between trypsin and the ligand was analyzed. It is found that the interactions between the ligand and the protein are dominated by van der Waals interactions and hydrogen bonding. Molecular dynamics (MD) simulations were used to verify the affinity between the ligand and trypsin; the simulations indicate that the complex remains stable, and the distance between the ligand and the target protein changes only a little. It is found that one water molecule acts as a bridge between the ligand and the protein pocket via hydrogen bonding. Final y, the ligand was coupled to Sepharose CL-6B gel, and was used to immobilize trypsin in an oriented fashion. It is found that the enzyme activity and specific activity of the oriented immobilized trypsin are 340.8 U·g-1 and 300.3 U·mg-1, respectively. These values are 10 and 5 times that of the free enzyme. The results of this work indicate that the combination of docking and MD simulations are promising for the rational design of ligands for oriented immobilization.% 在胰蛋白酶三维(3D)结构的基础上,首先利用分子对接从ZINC数据库中筛选获得了与胰蛋白酶具有较高亲和性的小分子配基2-硝基苯基-β-D-葡糖苷,并分析了该配基与蛋白质之间的相互作用力主要为范德华和氢键相互作用.并利用分子动力学模拟进一步验证了2-硝基苯基-β-D-葡糖苷与胰蛋白酶之间具有较强的亲和作用.分子动力学(MD)模拟结果表明,配基-目标蛋白质之间形成稳定的复合物且它们之间的距离基本没有变化.此外,一个水分子通过氢键在配基和目标蛋白质的结合腔之间架桥.最后制备了偶联有该配基的亲和载体,进行了胰蛋白酶的定向固定化,并考察了该固定化酶的活性.研究结果表明,利用修饰2-硝基苯基-β-D-葡糖苷配基的亲和载体固定化胰蛋白酶的酶活达到340.8 U·g-1,比活达到300.3 U·mg-1,分别是未修饰亲和配基载体的10倍和5倍,具有明显的优势.上述结论证明了结合分子对接和分子动力学模拟理性设计定向固定化亲和配基的方法是可行的,具有一定的理论和实用价值.
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