摘要：生物素是维持生物体内代谢平衡的重要物质，被广泛应用于医药、畜牧业、发酵工业等多个领域。生物素中间体单酯是生产生物素重要的中间体，我们课题组之前已经从巧克力微杆菌中找到可以水解生物素二甲酯为生物素单酯的特定酯酶，并在大肠杆菌中实现可溶性表达。本课题主要研究了酯酶与底物的结合模式，并对结合模式进行了分析，发现了与底物相互作用的三个氨基酸残基Ser(110),Asp(268)和His（330），并对活性中心附近43位的ASN、50位的TRP、203位的TRP、217位的TYR、227位的TRP，这5个氨基酸进行虚拟突变。突变之后的蛋白质序列又被用来分子对接，经分析后发现酯酶活性中心附近的氨基酸经突变后，使得底物酯酶间的结合能提高了，说明这些氨基酸残基的改变将会影响底物与酯酶之间的结合。47068

毕业论文关键词：巧克力微杆菌酯酶（EstSIT01）；分子对接；虚拟位点突变

Research on molecular docking and virtual site-directed mutagenesis of an Esterase from Microbacterium Chocolatum SIT01

Abstract： Biotin is an important material to maintain homeostasis in vivo, which is widely is widely used in medicine, animal husbandry and fermentation industries. Biotin intermediate monoester is an important intermediate for the production of biotin. In our previous study, we found that specific esterase from Microbacterium chocolatum could hydrolyze biotin-diester to become biotin monoester, and to achieve solubility in E. coli expression. In this paper, we researched and analyzed the binding mode between enzyme and substrate, and found three amino acid residues Ser (110), Asp (268) and His (330) which interact with the substrate. We made virtual mutation on five amino acids ASN(43), TRP(50), TRP(203), TYR(217) and TRP(227) near the active site of enzyme. Protein sequence mutated was used in molecular docking. It's got that the amino acid near the activity center of esterase mutated led to the increasement of binding energy needed in the combination of enzyme and substrate. It can indicate that the change of these amino acid residues in enzyme will affect the conjugation between enzyme and substrate.

Key Words: EstSIT01；Molecular Docking；Mutation

目录

1.前言 1

1.1开题依据 1

1.2生物催化 1

1.3酯酶 1

1.3.1酯酶 2

1.3.2酯酶的催化机理 3

1.3.3酯酶的应用 3

1.4分子对接 4

1.4.1分子对接简介 4

1.4.2分子对接研究进展 5

1.4.3 分子对接的应用 6

1.5生物素 7

1.6本课题意义及主要研究内容 7

1.6.1研究内容 7

1.6.2意义 8

2 实验部分 8

2.1实验软件 8

2.1.1Autodocking Tools 8

2.1.2Swiss-Pdbv 8

2.2实验方法 9

2.1.1分子对接 9

2.2.2虚拟位点突变 10

2.2.3 突变结果分析 11

3结果与讨论