基于仿生钛化和自组装的酶固定化载体设计与制备

发布时间：2018-02-24 17:04

本文关键词： 仿生钛化自组装氧化钛精蛋白酶载体包埋　出处：《天津大学》2009年博士论文　论文类型：学位论文

【摘要】： 仿生矿化过程可在温和条件下(常温、中性pH)利用生物模板或合成模板诱导无机氧化物的形成,并可通过模板的自组装来调控无机氧化物的形貌,这为酶固定化提供了新的途径。层层自组装过程可在常温水溶液中进行,条件温和,也能维持酶分子的天然构象和生物活性。这都将为固定化酶提供适宜的微环境。本论文主要研究仿生钛化和自组装及其用于固定化酶载体设计与制备。主要研究内容总结如下: 第一部分,仿生钛化过程机理的研究。利用精蛋白诱导钛的水溶性前驱体Ti-BALDH生成氧化钛纳米粒子,仿生合成的氧化钛为无定形的球形粒子,通过进一步高温煅烧处理可制得不同晶形的氧化钛。通过改变pH值、温度等条件可调控精蛋白自组装,进而调控氧化钛粒子大小。对仿生钛化过程机理及精蛋白在诱导合成氧化钛过程中的催化和模板双重作用进行了详细的阐述。第二部分,仿生钛化直接用于固定化醇脱氢酶(YADH)的研究。选用精蛋白作为诱导剂,Ti-BALDH作为钛前驱体,采用共沉淀法在精蛋白诱导生成氧化钛的同时将YADH固定,并提出了共沉淀法固定化酶的机理。与游离YADH相比,固定化的YADH能保持约95%以上的初始活性,并显示出良好的温度、pH、重复使用和储藏稳定性。第三部分,仿生钛化与LbL层层自组装相结合制备杂化微囊用于固定化YADH的研究。以CaCO3微粒为模板,在其表面交替组装精蛋白层和氧化钛层,然后用EDTA溶液去除模板得到protamine titania杂化微囊,阐明了微囊的形成过程和机理。将微囊用于固定化YADH的研究,发现YADH包埋率约为68%,微囊可为酶提供适宜的微环境,使酶保持较高的活性和重复使用稳定性。第四部分,仿生钛化用于制备多酶固定化载体。提出多酶微工厂的概念,设计并制备出具有Shells-in-Shell结构的多酶固定化载体。将protamine titania LbL层层自组装微囊包埋到Alg-Pro-Ti微囊中,以Alg-Pro-Ti微囊作为厂房, LbL微囊作为工作车间。含不同酶种的LbL微囊组成“酶催化装配线”,对输入的底物分子进行高效“加工”。Alg-Pro-Ti微囊将多酶共固定在局限空间内,缩短了多酶活性位点的距离,能提高反应的发生几率和效率,并有利于分离。最后,仿生钛化的拓展应用,利用仿生法制备氧化锆。以精蛋白和溶菌酶诱导仿生锆化过程,可得到无定形的氧化锆。详细探讨了溶菌酶在仿生锆化过程中的催化和模板双重作用,并对催化水解K2ZrF6的机理进行了解释。将仿生锆化过程用于YADH的固定化,固定化YADH表现出较高的温度和pH稳定性。
[Abstract]:Biomimetic mineralization can induce the formation of inorganic oxides under mild conditions (normal temperature, neutral pH) by using biological or synthetic templates, and the morphology of inorganic oxides can be regulated by self-assembly of templates. This provides a new way for enzyme immobilization. The layer by layer self-assembly process can be carried out in aqueous solution at room temperature under mild conditions. It can also maintain the natural conformation and biological activity of enzyme molecules, which will provide a suitable microenvironment for immobilized enzymes. In this thesis, bionic titanium and self-assembly and their applications in the design and preparation of immobilized enzyme carriers are studied. The main research contents are summarized as follows:. In the first part, the mechanism of bionic titanization was studied. The water-soluble precursor Ti-BALDH was induced to form titanium oxide nanoparticles by protamine, and the synthesized titanium oxide was amorphous spherical particles. Titanium oxide with different crystal shapes can be prepared by further calcination at high temperature. The protamine self-assembly can be controlled by changing pH value and temperature. The mechanism of bionic titaniation and the catalytic and templating roles of protamine in the induced synthesis of titanium oxide were discussed in detail. In the second part, the study of immobilized alcohol dehydrogenase (YADH) with bionic titanium was studied. The protamine was used as the inducer of Ti-BALDH as the precursor of titanium, and the YADH was immobilized by co-precipitation method while the protamine induced the formation of titanium oxide. The mechanism of immobilization of enzyme by coprecipitation method was put forward. Compared with free YADH, the immobilized YADH could maintain the initial activity of more than 95%, and showed good temperature pH, reuse and storage stability. In the third part, hybrid microcapsules were prepared by combining bionic titanium with LbL layer by layer self-assembly. Using CaCO3 particles as template, protamine layer and titanium oxide layer were alternately assembled on the surface. Then the protamine titania hybrid microcapsules were obtained by removing the template with EDTA solution, and the formation process and mechanism of the microcapsules were elucidated. When the microcapsules were used in the study of immobilized YADH, it was found that the embedding rate of YADH was about 68%, and the microcapsules could provide suitable microenvironment for the enzyme. The enzyme maintained high activity and reused stability. In 4th, bionic titanium was used to prepare multi-enzyme immobilized carrier. The concept of multi-enzyme microfactory was put forward, and the multi-enzyme immobilized carrier with Shells-in-Shell structure was designed and prepared. The protamine titania LbL was embedded in the Alg-Pro-Ti microcapsule layer by layer. Alg-Pro-Ti microcapsules were used as workshop and LbL microcapsules as workshop. LbL microcapsules containing different enzyme species constituted "enzyme catalytic assembly line". The imported substrate molecules were efficiently "processed". Alg-Pro-Ti microcapsules were co-immobilized in limited space. It can shorten the distance of multi-enzyme activity sites, improve the probability and efficiency of reaction, and is beneficial to separation. Finally, the application of bionic titanization is extended, and zirconia is prepared by bionic method. The process of bionic zirconization is induced by protamine and lysozyme. Amorphous zirconia can be obtained. The catalytic and template roles of lysozyme in bionic zirconization are discussed in detail, and the mechanism of catalytic hydrolysis of K2ZrF6 is explained. The bionic zirconization process is used for immobilization of YADH. The immobilized YADH exhibited high temperature and pH stability.
【学位授予单位】：天津大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R341;TQ11

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