Globo系列糖抗原Gb4的细胞工厂法克级合成  

Gram-scale Synthesis of Globo Series Carbohydrate Antigen Gb4 by Engineered Escherichia coli

作　　者：王晓雨 白静[1,2] 王志颖 周子璇 关婉怡 WANG Xiaoyu;BAI Jing;WANG Zhiying;ZHOU Zixuan;GUAN Wanyi(College of Life Sciences,Hebei Normal University,Hebei Shijiazhuang 050024,China;Department of Agriculture and Animal Husbandry Engineering,Cangzhou Technical College,Hebei Cangzhou 061001,China)

机构地区：[1]河北师范大学生命科学学院,河北石家庄050024 [2]沧州职业技术学院农牧工程系,河北沧州061001

出　　处：《河北师范大学学报（自然科学版）》2021年第5期512-521,共10页Journal of Hebei Normal University：Natural Science

基　　金：河北省引进留学人员资助项目(C20190348);河北省自然科学基金(C2020205049)。

摘　　要：Globo系列糖抗原Gb4广泛存在于多种细胞表面,参与细胞识别等多种生理活动,对机体生命活动起着重要作用.利用细胞工厂法大量合成Globo系列糖抗原Gb4,首先构建基因工程菌株,通过添加乳糖(Lac,二糖)为底物进行发酵,直接合成Gb4(四糖),并采用单因素分析法,依次对受体底物质量浓度、糖供体的前体糖的种类和质量浓度、发酵温度以及发酵时间进行了优化,最后确定1 g/L Lac为最适受体底物,添加5 g/L半乳糖(Gal)有利于糖供体UDP-Gal的合成,Gb4的合成产量提升了14倍,25℃为最适发酵温度,补加底物后21 h为最适发酵时间.以此最适条件在摇瓶进行大量发酵合成,胞内获得纯度约96%的Gb4,1 L发酵液可得到1.145 g的Gb4.该法简便迅速,极大地减小了寡糖合成的工作量,为Gb4的大量合成及功能研究奠定了基础.The Globo series carbohydrate antigen Gb4 is widely displayed on the surface of a variety of cells and participates in various physiological activities such as cell recognition,exhibiting important roles in biological events.In this study,the cell factory method is used for large-scale synthesis of Globo series carbohydrate antigen Gb4.First,genetically engineered strains are constructed,and lactose(Lac,disaccharide)is added as acceptor substrate during fermentation to directly synthesize Gb4(tetrasaccharide).Single factor analysis is employed to optimize the acceptor substrate concentration,the type and concentration of the precursor sugar of the sugar donor,the fermentation temperature and the fermentation time successively.The optimal Lac concentration is determined as 1 g/L,and addition of galactose(Gal)at 5 g/L can facilitate the synthesis of sugar donor UDP-Gal,resulting in a 14-fold increase in the yield of Gb4.The optimal fermentation temperature is determined as 25℃,and the optimal fermentation duration is 21 h after the supplementation of Lac and Gal.Then,a large-scale of flask fermentation is performed under the optimal condition,and Gb4 with a purity of about 96%is obtained from the cell.One liter of fermentation broth produces 1.145 g of Gb4.The method is simple and rapid,greatly reduces the workload of oligosaccharide synthesis,and thus lays a foundation for the large-scale synthesis and functional study of Gb4.

关 键 词：球形异四糖(Gb4) 细胞工厂法 单因素分析 

分 类 号：Q535.599[生物学—生物化学]