超大孔微球固定化酶体系的构建与应用

发布时间：2018-10-31 16:16

【摘要】：固定化酶是酶研究和应用中的重要内容,多孔微球以其高比表面积、可控的亲疏水性和功能基团成为优良的固定化酶载体,但常用的介孔微球会限制酶活性的发挥。本实验室前期的研究证明,超大孔聚苯乙烯(PST)微球对比介孔微球作为固定化脂肪酶载体,在酶活、重复使用性、稳定性以及动力学性质方面更有优势。这些令人感兴趣的结果促使本论文进行深入研究,探索载体孔径对固定化酶的影响机理,研究不同基质的超大孔微球固定化酶的效果,并探讨固定化酶的实际应用,初步考察载体孔径与酶分子大小的匹配关系。本论文主要分为四部分。(1)比较和研究脂肪酶在介孔微球、超大孔微球以及平面上的吸附状态,分析超大孔微球固定化酶具有优势的深层原因。通过对吸附时间和酶浓度的调控,阐明超大孔微球较低的孔曲率、较大的孔内空间、吸附酶的多层状态和更好的传质效果是其固定化酶比活和酶活较高的主要原因。脂肪酶吸附于平面时,由于没有孔结构的限制,酶活持续增长,比活基本保持不变。此外,底物浓度较高时,其富集作用会降低酶的催化效果。(2)比较不同基质的超大孔聚甲基丙烯酸缩水甘油脂微球(PGMA)、苯乙烯-甲基丙烯酸缩水甘油酯共聚微球(P(ST-co-GMA))和PST微球作为脂肪酶固定化载体的效果,并与商品化固定化酶Novozym 435对比。结果表明,超大孔PGMA微球固定化酶有更高的酶活、更好的重复使用性、稳定性和动力学性质等。考察共价结合以及载体亲疏水性对固定化酶性能的影响,通过吸附曲线、环氧基含量测定、接触角测定以及单独的物理吸附固定化酶等的考察,证明脂肪酶与载体的共价结合是PGMA微球具有显著优势的主要原因。(3)考察超大孔微球固定化脂肪酶催化棕榈酸异辛酯的合成反应,在最优的反应条件下,超大孔PGMA微球固定化酶催化反应的酯化率可达91.3%,重复使用10次后,其酯化率仍有87.1%。使用多次已失活的固定化脂肪酶加入PBS缓冲液可以部分恢复活性,超大孔PGMA微球固定化酶可以恢复至初始活力的80%以上。(4)将不同分子大小的酶固定于不同孔径、不同亲疏水性的微球,得到载体选择的规律,固定化酶载体的选择很复杂,要综合考虑孔径、亲疏水性、固定化方式以及底物传质等影响。大孔径的载体可以很好保持酶活；根据酶和底物的性质选择不同亲疏水性的载体；共价结合对于某些酶的活性和稳定性有提升。同时也得到酶分子大小与载体孔径间的匹配规律,酶分子尺寸与载体孔径并不一定是正相关的关系,还要考虑载量的影响和底物和产物的传质要求。综上所述,超大孔微球的结构、优良的传质性能和其中脂肪酶的多层吸附状态使其作为固定化酶载体具有优势,与共价结合法联合作用可以提高固定化脂肪酶的活性和稳定性,在实际应用中有较高的催化效率,通过对微球的调控,超大孔微球适用于多种类型酶的固定化,是一种理想的固定化酶载体。
[Abstract]:The immobilized enzyme is an important content in the research and application of enzyme. The porous micro-sphere is an immobilized enzyme carrier with its high specific surface area, controllable hydrophobicity and functional group, but the commonly used micro-spheres will limit the enzyme activity. It has been shown in the previous study that the micro-spheres of large pore polystyrene (PST) microspheres as immobilized lipase carriers have more advantages in enzyme activity, reusability, stability and dynamics. The results of these interesting results have led to an in-depth study on the effect of carrier pore size on immobilized enzyme, the effect of immobilized enzyme on large pores of different substrates, and the application of immobilized enzyme. The relationship between the pore size and the size of the enzyme was preliminarily investigated. The thesis is divided into four parts. (1) To compare and study the adsorption state of lipase on micro-sphere, ultra-large hole micro-sphere and plane, and to analyze the deep cause of micro-sphere immobilized enzyme. By controlling the adsorption time and the enzyme concentration, the pore curvature, the larger pore space, the multi-layer state of the adsorption enzyme and the better mass transfer effect of the super-large pore micro-sphere are explained. When the lipase is adsorbed on the plane, the enzyme activity continues to grow continuously because of the restriction of pore structure, and the specific activity remains unchanged. In addition, when the substrate concentration is high, its enrichment function reduces the catalytic effect of the enzyme. (2) To compare the effects of macropore polyglycidyl methacrylate (PGMA), styrene-glycidyl methacrylate copolymer microspheres (P (ST-co-GMA)) and PST microspheres as a lipase immobilization carrier in different substrates, and to compare with the commercial immobilized enzyme Novozym 435. The results showed that the immobilized enzyme of PGMA microspheres had higher enzyme activity, better reusability, stability and dynamic properties. The effects of covalent binding and hydrophobicity on the properties of immobilized enzyme were investigated. The adsorption curves, the determination of epoxy group content, the measurement of contact angle and the independent physical adsorption of immobilized enzyme were studied. It is proved that the covalent bond between lipase and carrier is the main reason for PGMA microspheres. (3) The synthesis reaction of the immobilized lipase catalyzed by the super-large pore microball immobilized lipase was studied. Under the optimum reaction conditions, the esterification rate of the immobilized enzyme catalyzed reaction of the super-large hole PGMA was 91.3%, and after 10 times of repeated use, the esterification rate of the immobilized lipase was 87. 1%. By adding the immobilized lipase to PBS buffer for partial recovery activity, the immobilized lipase with super-large pore PGMA can be recovered to more than 80% of the initial activity. and (4) fixing the enzyme with different molecular sizes to different pore diameters and different hydrophilic microspheres to obtain the law of carrier selection, and the selection of the immobilized enzyme carrier is complex, and the influence of pore size, hydrophobicity, immobilization mode and mass transfer of the substrate is comprehensively considered. the large pore size carrier can be well kept enzyme activity; different lipophilic carriers are selected according to the nature of the enzyme and the substrate; and covalent binding improves the activity and stability of certain enzymes. meanwhile, the matching between the size of the enzyme molecule and the pore diameter of the carrier is obtained, the size of the enzyme and the pore size of the carrier are not necessarily related, and the influence of the load and the mass transfer requirement of the substrate and the product are also considered. In conclusion, the structure of the super-large-hole micro-sphere, the excellent mass transfer performance and the multi-layer adsorption state of the lipase have the advantages as an immobilized enzyme carrier, and the activity and the stability of the immobilized lipase can be improved by the combination of covalent binding and the like, In practical application, there is a high catalytic efficiency, which is suitable for immobilization of various types of enzymes by controlling micro-spheres, which is an ideal immobilized enzyme carrier.
【学位授予单位】：中国科学院研究生院(过程工程研究所)
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q814.2

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