α半乳糖苷酶基因工程菌的开发.pdfVIP

α-半乳糖苷酶基因工程菌的开发 摘 要 α-半乳糖苷酶（α-Galactosidase,EC 2 ）, 是一种催化水解α-D-半乳糖苷( 半乳糖寡糖、半乳糖甘露聚糖)非还原性末端α-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α-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α-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 pPICz αA-AB、 pPICz αA-AC、pPICz αA-AA、pPICz αA-AZ 和 pPICz αA-AF 五种 α-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α-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 α-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 α-半乳糖苷酶的工业化生产和 应用奠定了基础。 关键词： α-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 α-galactosidase producing strain Abstract α-Galactosidase (EC 2), is one kind of enzyme that hydrolyze non-reducing terminal α-D- galactosidic bonds in α-D-galactoside. The enzyme is currently widely used in food and feed industry. However, due to the low production capacity and high cost of α-galactosidase, it cannot meet the needs of industrial applications. In order to reduce the producing cost,this research aims to construct engineered Pichia pastoris strain with high level secretory expression of α-galactosidase through molecular biology. In this research,five α-galactosidases (AB, AC, AA, AZ and AF) from different species were investigated and their expression vectors were constructed, named pPICz αA-AB, pPICz αA- AC, pPICz αA-AA, pPICz αA-AZ and pPICz αA-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 expression of α-galactosidases including increasing gene copy number and co-expressing of chaperone HAC1. T