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重组鼠李糖苷酶的表面展示酿酒酵母产酶及其催化特性

时间:2023-01-26 21:39来源:毕业论文
重组鼠李糖苷酶的表面展示酿酒酵母产酶及其催化特性.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-

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

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