《酿酒酵母碱性蛋白酶表达载体的构建及表达状况的研究》-毕业论文.doc

编号 本科生毕业设计（论文） 题目： 酿酒酵母碱性蛋白酶表达载体的构建及表达状况的研究 生物工程学院 发酵 专业 学 号 学生姓名 指导教师 教授 副教授 二〇一一年六月 摘 要 随着石化燃料的日益减少，酒精作为一种清洁可再生的新型能源成为当前各国研究的热点。酿酒酵母酒精发酵是目前工业上最大的、应用最广的生物乙醇生产方式。因此，有效地降低生产成本是目前研究者研究的热点。利用基因工程及代谢工程技术对工业酒精酵母进行改造，可提高菌种的发酵性能，降低酒精的生产成本。 目前生产酒精的主要原料，淀粉质原料如玉米，饲用大麦等含有较高浓度的蛋白质(玉米: 10-13%; 饲用大麦: 15-18 %)。而酿酒酵母无外分泌蛋白酶，不能利用原料中的可溶性蛋白。为使酿酒酵母能够利用原料中的可溶性蛋白，提高酒精产率和原料利用率，降低酒精发酵的成本，本实验通过信号肽分泌表达和酿酒酵母表面展示技术并以酿酒酵母自主复制和同源重组两种不同的方式表达来源于枯草芽孢杆菌（Bacillus subtilis168）的碱性蛋白酶基因，获得表面展示表达重组菌S.C.pMD1 (pMD-PGK1-α-factor-AG1)[整合型] 、S.C.pYX1(PYX212-KAN-α-factor-AG1)[游离型]、及信号肽分泌表达重组菌S.C.PMD2 (PMD-PGK1-α-factor)[整合型] 、S.C.PYX2(PYX212-KAN-α-factor)[游离型]。在重组菌碱性蛋白酶酶活测定的实验中，重组子都表达出一定量的酶活。但总体而言，游离型表达质粒的酶活表达量要高于整合型表达质粒。由于时间关系，还没有对信号肽分泌表达和表面展示表达做出相应的酶活测定和分析。 关键字：碱性蛋白酶；酿酒酵母；酒精发酵；表面呈现表达 ABSTRACT As the reserves of petroleum decrease, ethanol, which is renewable, has become the focus of many countries as an alternative liquid fuels to gasoline. Bioethanol production by saccharomyces cerevisiae is currently by volume, the single lagest fermentation process in industrial biotechnology. Therefore the development of cost-effective technologies for fuel ethanol production is a priority for many researchers. Improvement of the fermentation competence of yeast strain by gene engineering and metabolic engineering will decrease the costs of ethanol production. Although the materials used for ethanol production contain relatively high protein content (corn: 10-13%; feeding barley: 15-18 %), the yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. To improve the substrate utilization and ethanol yield, the Apr E gene ,encoding alkali protease Bacillus subtilis 168, was cloned and expressed in industrial ethanol yeast by means of many expressing techniques. In this experiment,we used the signal peptide secretion and wine yeast surface display technology as well as wine yeast independent copy and homologous recombination of