摘 要：酯酶是一类催化酯键的水解和形成的酶。他们现在已经成为制药，食品和生物降解应用的重要生物催化剂。在以前的工作中，实验室筛选出巧克力微杆菌SIT 101，其产生相应的（4S，5R）构型的高度立体选择性催化的外消旋生物素中间体二甲酯不对称水解，其是合成D-生物素。 本毕业论文主要通过优化培养基组成和发酵条件，提高重组菌的活性，为工业应用奠定基础。分别优化了碳源和碳源浓度、氮源和氮源浓度、金属离子和金属离子浓度、诱导剂浓度和诱导时间8个单因素，最终优化出理想的培养基成分和发酵条件，实现高的细胞量和高酶活这一目标，为工业应用奠定基础。最终优化出的培养基组分为：甘油（30 g/L）、酵母浸膏（20 g/L）、NaCl （5 g/L）、Fe2+（9 mM）；优化出的条件为：在培养3h后加入终浓度为0。6 mM的IPTG，发酵液初始pH7。4。将优化前后的培养基做了对比实验发现，优化后的细胞量和酶活力分别提高了2。5倍和10倍。这说明优化出的培养基极大地促进重组菌生长和产酶。此外，本文还利用蛋白质三级结构预测方法的同源建模预测重组酯酶EstSIT01的蛋白质三级结构，并对其进行了评价和优化。81286

毕业论文关键词：酯酶；D-生物素；发酵优化；同源建模

Fermentation optimization, molecular simulation of recombinant esterase

Abstract：Esterases are enzymes which could hydrolyze or form ester bonds, have been extensively applied in various areas as an important biocatalyst, especially in the food, pharmaceutical industry and environmental bioremediation。In our previous study, Microbacterium chocolatum SIT 101 was screened out for the conversion of prochiral diester to enantiomerically enriched (4S,5R)-monoester [(4S,5R)-2-imidazolone-4,5-dicarboxylic acid mono-ester], which is an important intermediate of D-biotin。 D-biotin is an important water-soluble B vitamins。 This thesis mainly by optimizing the composition and fermentation conditions of the culture medium and improving the activity of recombinant bacteria, thus laying the foundation for industrial application。 Respectively to optimize the concentration of carbon source and carbon source, nitrogen source and nitrogen source concentration and the concentration of metal ions and metal ions, concentration of inducer and the induction time eight single factor and eventually optimize the culture medium and fermentation conditions for ideal, to achieve high cell volume, and the goal of high enzyme activity, lay the foundation for the industrial application。 The resulting cultures were pided into: glycerin (30 g/L), yeast extract (20 g/L), NaCl (5 g/L), Fe2 + (9 mM)。 The optimum condition is: IPTG, which is 0。6mm after the culture of 3h, and the initial pH7。4 in fermentation。 Compared with the research of the culture medium before and after optimization, the improved cell volume and enzyme activity increased by 2。5 times and 10 times respectively。 This shows that the optimized culture promotes the growth and enzymes of recombinant bacteria。 The graduation design mainly through the optimization of medium composition and fermentation conditions to improve the activity of recombinant bacteria, lay the foundation for industrial applications。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: esterases ;D-Biotin; fermentation optimization ; protein structure

目录

1  研究背景 1

 1。1 酯酶 1

  1。1。1 酯酶的简介 1

  1。1。2 酯酶的立体结构和催化机制 1

  1。1。3 酯酶的应用