CAS OpenIR
基于微流控技术酶法合成6‘-唾液酸乳糖胺的初探
吴丽娜
Thesis Advisor杜昱光, 赵勇
2020-07-01
Degree Grantor中国科学院大学
Degree Name硕士
Degree Discipline生物工程
Keyword寡糖合成,糖-核苷酸的生物合成,糖基转移酶,生物催化,微流控技术
Abstract

糖链对糖复合物发挥正确功能有着非常关键的作用,作为探索生命奥秘的第三个里程碑,糖链研究已经成为继蛋白质研究与核酸研究后的重要研究领域。基于糖链具有结构复杂性和多样性的特点,通过纯化获取糖链获仍具有一定难度,获得特定糖链的方式仍然是化学法或生物法合成。由于受限于糖链的获得,许多糖链的结构和功能之间关系至今仍然未知。因此,发展新型、环保、经济、高效的糖链合成技术及研发糖链自动合成仪,具有十分重要的理论研究意义和实际应用价值。本研究基于微流控平台的酶催化合成糖链技术开展了以下研究:(1)以N-乙酰-α-2,6-唾液酸乳糖胺的酶催化合成为例,先合成生物素连接的底物(即 GlcNAc-Biotin I),再酶法合成生物素标记的N-乙酰-α-2,6-唾液酸乳糖胺,完成模块反应及反应条件优化,第一步反应率90.00 % 转化率,第二步反应率实现95.00 % 转化率。以磁珠为固相支持物的酶催化合成反应,实现反应的固相合成及优化反应条件。集成数字微流控、固相合成及酶催化模块反应等合成糖链技术,优化改进合成方式及条件。(2)以GlcNAc-Biotin II为底物,提高反应效率的同时增加光敏基团,实现更加高效环保的合成过程,优化第一步反应率提高到93.70 %,第二步反应率提高到97.28 %。(3)实现超高频声微流控微流控、固相纯化技术以及酶催化模块合成糖链等技术集成,并成功在该平台上连续反应获得产物,使得芯片上第一步反应实现78.00 %的转化率,第二步反应实现91.70 %的转化率,连续反应可以成功的合成并被检测到。为发展一种高效、经济、绿色的自动糖链合成技术奠定基础。;Glycans of glycoconjugates such as glycoproteins and glycolipids are very important for their proper functions. Glycans research has become the third milestone in exploring the mysteries of life after protein and nucleic acid research. Due to the diversity and complexity of glycans structure, obtaining glycans through purification is still challenging. At present, the best choice for obtaining glycans is still the method of synthesis. Therefore, the development of innovative, environmentally friendly, economical and efficient glycans synthesis technology has important theoretical research on the structure-function relationship of glycans and the development of glycans synthesis instruments. Significance and practical application value. This research will aim to develop an enzyme-catalyzed glycans synthesis technology based on a microfluidic platform, We carried out the following research: (1) Take the enzyme-catalyzed synthesis of N-acetyl-α-2,6-sialyllactosamine as an example, first synthesize avidin-linked substrate (GlcNAc-Biotin I), and then enzymatically synthesize N-acetyl- α-2,6-Sialyl lactosamine reaction module. Optimize the conditions of the module so that the conversion rate in the first step reaches 90.00% and the conversion rate in the second step reaches 95.00%. The enzyme-catalyzed synthesis reaction using magnetic beads as a solid support supports the solid-phase synthesis of the reaction and optimizes the reaction conditions. Integrate digital microfluidics, solid-phase synthesis and enzyme-catalyzed modular reactions to synthesize Glycans technologies to optimize and improve synthesis methods and conditions. (2) Using GlcNAc-Biotin II as a substrate to increase the reaction efficiency while adding photosensitive groups to achieve a more efficient and environmentally friendly synthesis process, the optimization of the first step reaction rate increased to 93.70%, the second step reaction rate increased to 97.28% . (3) Achieve ultra-high frequency acoustic microfluidic microfluidic, solid phase purification technology and enzyme catalytic module synthesis of sugar chains and other technical integration, and successfully continuous reaction on the platform to obtain products. The first-step reaction on the chip achieves a conversion rate of 78.00% and the second-step reaction achieves a conversion rate of 91.70%. Continuous reactions can be successfully synthesized and detected. It lays the foundation for the development of an efficient, economical and green automatic Glycans synthesis technology. 

Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/49723
Collection中国科学院过程工程研究所
Recommended Citation
GB/T 7714
吴丽娜. 基于微流控技术酶法合成6‘-唾液酸乳糖胺的初探[D]. 中国科学院大学,2020.
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