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题名	磁性纳米颗粒强化多酶偶联催化体系在生物合成中的应用
作者	郑木青
学位类别	博士
答辩日期	2011-08-16
授予单位	中国科学院研究生院
导师	王平
关键词	多酶偶联反应 磁性纳米颗粒 辅因子再生 固定化
其他题名	Multi-enzyme catalytic system intensified by magnetic nanoparticles for biosynthesis
学位专业	生物化工
中文摘要	随着工业生物技术的发展，由固定化的多酶体系尤其是包含辅因子再生的体系实现多步反应，已经引起了越来越多的关注。但是，在固定化的多酶体系中实现辅因子有效的再生仍然是一个具有挑战性的课题。针对目前多酶偶联体系固定化技术存在的问题，本论文利用超顺磁性的二氧化硅纳米颗粒进行酶和辅因子的固定化，旨在从内在（固定化模式、间隔臂）以及外在（固定化多酶偶联反应的体系环境）两个方面，系统的研究影响多酶体系动力学的因素，从而实现多酶偶联体系的高效催化。在前人工作的基础上，优化反应条件制备得到平均粒径为124 nm的磁性二氧化硅纳米颗粒。所得纳米颗粒尺寸均一、性质稳定、在水中能够单分散、并且具有超顺磁性。为了证实所制备的磁性二氧化硅纳米颗粒固定化酶的可行性和有效性，首先进行了甘油脱氢酶的固定化，并催化甘油转化为1,3-二羟基丙酮。结果表明，固定化甘油脱氢酶具有57.5 mg/g的高负载量、81.1%的高活性收率，提高了对温度和pH变化的耐受性、热稳定性，并且具有良好的重复使用性。甘油脱氢酶固定化的结果充分表明，制备的磁性二氧化硅纳米颗粒可以作为固定化酶的理想载体。构建了由谷氨酸脱氢酶（Glutamate dehydrogenase，GluDH），葡萄糖脱氢酶（Glucose dehydrogenase，GDH），和辅因子烟酰胺腺嘌呤二核苷酸（NAD+/NADH）组成的多酶偶联体系，以实现辅因子的再生。采用将两种酶和辅因子分别固定化在纳米颗粒上（E-E-C）、将两种酶共固定化在同一纳米颗粒上而辅因子单独固定化(EE-C)，以及酶和辅因子集成固定化在同一纳米颗粒上（EEC）三种模式将多酶体系固定化在磁性纳米颗粒上。结果表明，分别固定化在不同纳米颗粒上的多酶也可以通过纳米颗粒的之间的碰撞实现偶联反应。与E-E-C固定化模式相比，EEC体系具有更高的催化效率，是分别固定化体系的1.8倍左右。另外，还发现在酶和辅因子固定化之前连接较长的间隔臂可以将E-E-C体系的催化效率提高2倍左右，但对于EEC体系的催化效率影响微弱。进一步通过改变固定化多酶偶联反应体系的环境，考察影响E-E-C多酶偶联体系催化效率的因素。将交变磁场（500 Hz，14.2 Gs）施加于E-E-C多酶偶联体系，结果发现，偶联反应的速率提高至2.3倍，这证实了交变磁场能够强化超顺磁性纳米颗粒的运动性，促进酶和辅因子之间的相互作用，进而提高偶联反应的速率。提高外加交变磁场的强度和频率均对多酶偶联反应的催化效率产生促进作用。另外，在一定范围内，偶联反应速率随着反应体系粘度的增大而降低。通过本文的研究，为进一步阐释影响包括辅因子再生的固定化多酶偶联体系的分子运动机制提供了实验和理论依据，为研制更高效的多酶催化剂并利用其进行高效的多酶偶联反应提供了理论指导。
英文摘要	With the development of industrial biosynthetic technology, multistep biotransformations realized with immobilized multi-enzymatic systems especially those involving cofactor regeneration have attracted more and more attentions. However, efficient cofactor regeneration in immobilized multienzyme catalysis remains a big challenge. Aiming at the difficulties faced in immobilized multi-enzymatic system, this work applied superparamagnetic silica nanoparticles to immobilize enzymes and cofactor. Internal and external factors affecting immobilized multi-enzymatic reaction kinetics were systematically investigated to facilitate the high efficiency biotransformation of coupled multi-enzymatic system. Magnetic silica nanoparticles were synthesized using a modified method based on the work previously reported. It was found that the prepared silica magnetic nanoparticles have a high uniform size of 124 nm and good dispersibility in water, and they also exhibited superparamagnetism. To identify the feasibility and effectivity for enzyme immobilization, the prepared magnetic nanoparticles were applied for glycerol dehydrogenase (GlyDH) immobilization to catalyze the concersion of glycerol to 1,3-dihydroxyacetone. It showed that immobilized GlyDH achieved high enzyme loading of 57.5 mg/g with high activity recovery of 81.1%. Moreover, increased the tolerance to changes in pH and temperature, and showed improvement in thermal stability and excellent reusability. The result indicated that the prepared magnetic nanoparticles could be used as ideal carriers for enzyme immobilization. A multi-enzymatic system including glutamate dehydrogenase (GluDH), glucose dehydrogenase (GDH) and NADH was constructed, and the synthesized magnetic nanoparticles were applied to immobilize the enzymes and cofactor. Three immolibzation patterns were tested: separately immobilized enzymes and cofactor (labeled as E-E-C), coimmobilized enzymes with separately immobilized cofactor (EE-C), and coimmobilized enzymes and cofactor (EEC). It showed that particle collision of nanoparticles successfully enabled the coupled reactions involving the regeneration of cofactor even when the enzymes and cofactor were immobilized separately on nanoparticles. Compared with E-E-C system, EEC immobilization had higher catalysis efficiency and could enhance the reaction rate by a factor of 1.8. It was further observed that the use of spacer could also double the reaction rate of the E-E-C system, with little effect on the EEC system. For E-E-C system, other factors were further investigated by manipulating the environment where the multi-enzymatic reaction took palce. It was found that when an alternating magnetic field (500 Hz, 14.2 Gs) was applied, the reaction rate increased to about to 2.3 times of that without magnetic field. The result confirmed that the magnetic field could enhance the mobility of nanoparticles, promote the interactions between enzymes and cofactor, thus led to the increase of reaction rate. Increase both in magnetic field intensity and frequency also led to the increase in reaction rate. Further, viscosity of reaction solution also had effect on the reaction. With the increase of reaction solution viscosity, the reaction rate of the multi-enzyematic reaction decreased. The present study will be helpful for a better understanding on the factors affecting the molecular mobility of multi-enzymatic system involving cofactor regeneration, and will provide both experimental and theoretical guidance for preparation and utilization of highly efficient multi-enzymatic biocatalysts and complex biotransformations.
语种	中文
公开日期	2013-09-24
内容类型	学位论文
源URL	[http://ir.ipe.ac.cn/handle/122111/1759]
专题	过程工程研究所_研究所（批量导入）
推荐引用方式 GB/T 7714	郑木青. 磁性纳米颗粒强化多酶偶联催化体系在生物合成中的应用[D]. 中国科学院研究生院. 2011.

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