摘 要：α-生物素又名维生素H，在维持人和动物正常生长发育等方面起着重要作用，在医药和饲料等行业被广泛应用。生物素手性单甲酯是合成生物素的关键手性中间体。生物催化具有反应条件温和、高度选择性、产物纯度高等优点。本毕业设计主要利用高效液相色谱法的分析方法针对该酶催化水解反应的反应条件进行优化，主要包括水溶性有机助溶剂和非水溶性有机助溶剂的影响，分散剂的影响以及乙二胺四乙酸二钠盐的影响等等，由实验结果可知非水溶性有机助溶剂甲苯对底物的溶解能力较强，并且能够很大程度地提高该反应的转化率，是比较理想的助溶剂。另外，该论文还利用同源建模的蛋白质三级结构预测方法对重组酯酶EstSIT01的蛋白质三级结构结构作预测以及评估和优化。62006

毕业论文关键词： α-生物素；生物催化；同源建模

The biological simulation of recombinant esterase and the optimizing of the enzymatic hydrolysis reaction of biotin dimethyl

Abstract：α-Biotin , which also called Vitamin H, it plays an remarkably important role on maintaining normal growth of humans and animals.It’s also widely used in the field of pharmaceutical and feed. Optical lactone cis-1,3-dibenzyl—hexahydrofuro[3，4-d]imidazole-2,4-dione is the key chiral intermediate to synthesis biotin.Biocatalysis owns many advantages such as mild reaction conditions, high selectivity and product of high purity. The main content of this graduation project is optimizing the reaction conditions of the enzymatic hydrolysis reaction by HPLC.It includes the effect of water-soluble organic cosolvent , water-insoluble organic cosolvent , dispersant and EDTA-2Na (to add the permeability of the cell),and so on.From the experimental results, we can find that Toluene which is one of the water-insoluble organic cosolvent owns better solubility of the substrate and improves the conversion rate highly.So, toluene is an ideal cosolvent of this reaction. In addition, this paper also studies the protein structure of this enzyme and hope to predict and optimize it’s structure by homology modeling.

KeyWords: α-Biotin; biocatalysis ; protein structure

目录

1  研究背景 1

 1.1 生物素及其合成方法的研究 1

  1.1.1 生物素的性质及作用 1

  1.1.2 生物素的生物合成方法 1

 1.2 酶催化法合成生物素手性中间体内酯 3

  1.2.1 重组大肠杆菌在生物素手性中间体内酯合成中的作用 3

  1.2.2 水解酶的性质以及酶在手性合成中的作用 4

 1.3 蛋白质三级结构同源建模的原理和意义 5

  1.3.1 蛋白质三级结构预测 6

  1.3.2三种主要的蛋白质结构预测方法 6

 1.4 本课题研究意义和思路 7

  1.4.1 研究意义 8

  1.4.2 研究思路 8

2  实验原理和实验流程 8

 2.1 实验原理 8

  2.1.1酶催化法合成手性中间体 8

  2.1.2 同源建模的原理 9

3  试验方法与材料 10

 3.1 实验材料