1、-半乳糖苷酶基因工程菌的开发学 院：专 业：姓 名：指导老师：材料与环境学院生物工程邓智远学 号：职 称：160504104076刘小刚 梁伟凡讲师 高级工程师中国珠海二二年五月北京理工大学珠海学院16届本科生毕业论文诚信承诺书本人郑重承诺：本人承诺呈交的毕业论文-半乳糖苷酶基因工程菌的开发是在指导教师的指导下，独立开展研究取得的成果，文中引用他人的观点和材料，均在文后按顺序列出其参考文献，设计使用的数据真实可靠。本人签名： 日期： 年 月 日-半乳糖苷酶基因工程菌的开发摘 要-半乳糖苷酶（-Galactosidase,EC 3.2.1.22）, 是一种催化水解-D-半乳糖苷(如半乳糖寡糖、半
2、乳糖甘露聚糖)非还原性末端-D-半乳糖苷键的酶。此酶在食品以及饲料方面大量运用。但是由于目前-半乳糖苷酶的生产水平较低，成本较高，满足不了工业应用上的需求，所以本论文旨在通过分子生物学技术构建高效表达-半乳糖苷酶基因工程菌，降低生产成本，为工业应用奠定基础。本论文调研了AB、AC、AA、AZ和AF五种不同来源的基因，并构建了pPICzA-AB、pPICzA-AC、pPICzA-AA、pPICzA-AZ和pPICzA-AF五种-半乳糖苷酶重组表达质粒，通过筛选比较， AA基因的酶活最高，50L发酵罐酶活达到912U/mL。后续在AA菌株的基础上，通过增加AA基因的拷贝数以及共表达HAC1分子伴侣
3、蛋白两种方式继续提高AA菌株的酶表达能力，结果共表达分子伴侣的菌株酶活大幅提高，其发酵液酶活高达4663U/mL，是AA菌株的5.1倍，也是目前公开报道的最高水平。本研究从-半乳糖苷酶高酶活基因筛选和蛋白高效表达策略两方面入手，构建了高效分泌表达-半乳糖苷酶的毕赤酵母工程菌，其发酵161小时的酶活达到4663U/mL，是目前公开报道的最高水平，本研究工作为-半乳糖苷酶的工业化生产和应用奠定了基础。关键词：-半乳糖苷酶；巴斯德毕赤酵母表达系统；基因筛选；蛋白高效表达Development of -galactosidase producing strainAbstract-Galactosida
4、se (EC 3.2.1.22), is one kind of enzyme that hydrolyze non-reducing terminal -D- galactosidic bonds in -D-galactoside. The enzyme is currently widely used in food and feed industry. However, due to the low production capacity and high cost of -galactosidase, it cannot meet the needs of industrial ap
5、plications. In order to reduce the producing cost,this research aims to construct engineered Pichia pastoris strain with high level secretory expression of -galactosidase through molecular biology. In this research,five -galactosidases (AB, AC, AA, AZ and AF) from different species were investigated
6、 and their expression vectors were constructed, named pPICzA-AB, pPICzA-AC, pPICzA-AA, pPICzA-AZ and pPICzA-AF, respectively. Among these genes, AA strain showed the highest activity and the activity of the fermentation broth reached to 912U/mL. Subsequently, two methods were applied to improve the
7、expression of -galactosidases including increasing gene copy number and co-expressing of chaperone HAC1. The result showed that co-expression of HAC1 had obvious improvement on -galactosidases expression while increasing gene copy number had no influence. The activity of AA/HAC1 strain was up to 466
8、3U/mL in 161h, which was 5.1 times compared to AA strain and was the highest productivity of -galactosidases up to date.In this work, gene screening and protein high level expression strategy were applied to construct engineered P.pastoris strain with high level secretory expression of -galactosidas
9、e. The activity of fermentation broth was up to 4663U/mL, the highest productivity up to date. This work lays the foundation of the industrial production and application for -galactosidase.Keywords：-galactosidase; Pichia pastoris expression system; gene screening; high protein expression目录1 前言11.1 -
10、半乳糖苷酶11.1.1 -半乳糖苷酶简介11.1.2 -半乳糖苷酶的结构与催化机制11.1.3 -半乳糖苷酶的应用21.1.4 -半乳糖苷酶基因工程领域的研究进展31.2 巴斯德毕赤酵母表达系统41.2.1 巴斯德毕赤酵母表达系统优势41.2.2 影响毕赤酵母中高效表达外源基因的因素41.2.2.1 基因结构41.2.2.2 基因拷贝数51.2.2.3 蛋白的分泌51.2.2.4 其他因素61.3 研究目的与思路62 实验材料与方法82.1 实验材料82.1.1 菌株和质粒82.1.2 试剂82.1.3 溶液和培养基82.1.4 实验仪器92.2 实验方法102.2.1 分子克隆基本操作102
11、.2.1.1 PCR102.2.1.2 核酸电泳112.2.1.3 基因回收112.2.1.4 质粒提取122.2.1.5 酶切132.2.2 表达载体构建132.2.2.1 连接132.2.2.2 转化142.2.2.3 菌落PCR验证142.2.3 感受态制备与转化152.2.3.1 感受态的制备152.2.3.2 毕赤酵母的转化162.2.4 菌种培养筛选162.2.5 酶活检测162.2.6 50L发酵罐培养193 实验数据与分析203.1 基因筛选与性能评价203.1.1 重组表达载体设计以及构建203.1.2 重组毕赤酵母菌株转化313.1.3 基因筛选313.1.3.1 孔板初筛
12、313.1.3.2 摇瓶验证343.1.4 发酵罐培养验证353.2 酶的表达优化363.2.1 增加基因拷贝数363.2.1.1 多拷贝重组表达载体的设计以及构建363.2.1.2 重组毕赤酵母菌株转化383.2.1.3 孔板筛选383.2.2 共表达分子伴侣蛋白383.2.2.1分子伴侣蛋白重组表达载体设计以及构建383.2.2.2 重组毕赤酵母菌株转化403.2.2.3 孔板筛选验证413.2.3 摇瓶验证413.3 发酵罐性能评价424 结论与建议44参考文献45谢辞47附录481 前言1.1 -半乳糖苷酶1.1.%莠欀瀀栀琀洀氀重倀儀5Smwap前台访问/p-2945968.html
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