摘要α-L-鼠李糖苷酶是一种工业水解酶，可以催化水解α-1, 2、α-1, 3、α-1, 4、α-1, 6及α1等糖苷键，广泛运用于糖苷类化合物的水解。然而，自然界中的α-L-鼠李糖苷酶分离纯化难度较大，市场上纯酶价格昂贵，商业化程度不高，因此亟需寻找一种新型制备α-L-鼠李糖苷酶的方法。本文探究了采用α凝集素作为载体蛋白，将α-L-鼠李糖苷酶展示在酿酒酵母细胞的表面，开发新型的全细胞催化剂。87209
以pYD1为载体构建含有α-L-鼠李糖苷酶基因（rhA）的重组质粒pYD1-rhA，以EBY100酿酒酵母作为受体菌株，构建重组酿酒酵母菌株EBY100-pYD1-rhA。通过MD平板筛选，获得表面展示型的RHA酵母工程菌株。重组酵母在YNB-葡萄糖液体培养基上培养12～16 h，更换至YNB-半乳糖液体培养基中，20 ℃诱导表达48 h，蛋白表达量最高，为0。78 μg/mL。
通过酶活检测，发现重组酿酒酵母菌株展示α-L-鼠李糖苷酶的最适温度及最适pH值分别为70 ℃和pH 5。0。以制备的全细胞催化剂催化芦丁水解生成异槲皮苷，其转化率为9。8 %，无副产物槲皮素生成。因此，构建的酿酒酵母展示的α-L-鼠李糖苷酶具有良好的催化立体选择性，可作为一种有潜在商业用途的催化剂。
毕业论文关键词：α-L-鼠李糖苷酶； 细胞表面展示； 酿酒酵母（Saccharomyces Cerevisiae）； 异槲糖苷；生物转化
Abstract α-L-rhamnosidase as a sort of hydrolytic enzyme was widely applied to catalysis the hydrolysis of glycosidic bonds, including α-1，2、α-1，3、α-1，4、α-1，6 and α1。 However, it was difficult to separate and purify it from nature。 In addition, not only the price of pure α-L-rhamnosidase is very high, but the level of commericial utilization is low in the market。 Therefore, it is urgent to find a novel method to prepare α-L-rhamnosidase。 In the present study, in order to explore the novel whole-cell biocatalyst, α-lectin was used as a carrier protein to display the α-L-rhamnosidase on the surface of yeast cell。
Plasmid pYD1 was used as vector to construct the exoression plasmid pYD1-rhA。 Then, the exoression plasmid pYD1-rhA was transformed into Saccharomyces cerevisiae EBY100 to construct recombinant strain Sccharomyces cerevisiae EBY100-pYD1-rhA。 The RHA expressing yeast strains were obtained by minimal dextrose (MD) plates。 The EBY100-pYD1-rhA was cultured in YNB-glucose liquid medium for 12～16 h。 The cells were harvested and resuspended in YNB-galactose liquid culture medium, then induced at 20 ℃ for 48 h, and the content of α-L-rhamnosidase was 0。78 μg/mL。
By enzyme activity assay, the optimum temperature and pH of α-L-rhamnosidase was 70 ℃ and pH 5。0, respectively。 Hydrolysis of rutin was performed by adding RHA display on yeast cell, and 9。8 % isoquercitrin was obtained and without by-products quercetin。 The results indicated that α-L-rhamnosidase displayed by Sccharomyces cerevisiae exhibited significant stereoselectivity in the biological transformation of rutin。 Therefore, the surface display of α-L-rhamnosidase showed potential applications in industrial fields。
Keywords: α-L-Rhamnosidase; Cell surface display; Saccharomyces Cerevisiae; Isoquercitrin; Biocatalysis and biotransformation。
目 录
第一章 绪论 1
1。1 酶与生物催化 1
1。1。1 生物催化与转化 1
1。1。2 生物催化剂 2
1。2 鼠李糖苷酶 4
1。2。1 鼠李糖苷酶的基因型 重组鼠李糖苷酶的表面展示酿酒酵母产酶及其催化特性:http://www.youerw.com/shengwu/lunwen_129390.html