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超大孔微球固定化酶体系的构建与应用
王伟宸
学位类型博士
导师马光辉
2016-07
学位授予单位中国科学院研究生院
学位授予地点北京
学位专业生物化工
关键词超大孔微球 固定化 脂肪酶 孔径 共价结合
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)微球与介孔微球作为固定化酶载体来比较,在固定化酶的活性保留、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可以保持更好催化效果的主要原因。脂肪酶吸附于平面时,多层吸附也可以促进酶活保持,但吸附状态与酶在孔内的吸附不同,吸附量呈线性增长,酶的比活几乎不变。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))以及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。结果显示,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,通过一系列表征,包括吸附曲线、环氧基含量测定、接触角测定以及物理吸附固定化酶等,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微球固定化酶催化的酯化率可达91.3%。使用多次已失活的固定化脂肪酶加入PBS可以复性,超大孔PGMA微球固定化酶可以恢复至初始活力的80%以上。(4) 采用不同分子大小的酶固定于不同孔径、不同亲疏水性的微球,得到酶分子大小与载体孔径间的匹配规律。对于淀粉酶(2~3 nm),大孔径载体中固定化效果更好。对于葡萄糖氧化酶(8.4 nm),适于吸附在亲水性载体或固定于PGMA微球,孔径为100~200 nm的微球使其酶活较高。对于过氧化氢酶(10.4 nm),孔径为100~200 nm的PGMA微球较适宜其固定化。大孔径的载体对于固定化酶有更佳的效果,共价结合、载量都会影响酶的活性保持。综上所述,超大孔微球的结构、优良的传质性能和其中酶的多层吸附状态使其在固定化酶方面具有优势,与共价结合法联合作用可以提高固定化酶的活性和稳定性,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酶的固定化,是一种理想的固定化酶载体。

其他摘要

Immobilized enzyme is an important part of research and application of enzyme. Porous microspheres are fine carriers for enzyme immobilization because of their advantage of high specific surface area, controllable hydrophilicity/hydrophobicity and functional groups. In the meantime, frequently-used mesoporous microspheres might limit the catalytic performance of enzyme due to the high mass transfer resistance in the small pores. In the previous studies of our laboratory, the gigaporous polystyrene (PST) microspheres were used to immobilize lipase and the activity preservation, stability and kinetic properties of immobilized lipase were superior to those of mesoporous ones. In this study, the reasons of advantages of gigaporous microspheres as immobilization carriers were analyzed as well as the mechanism of pore size influencing enzyme immobilization. The gigaporous microspheres with different surface groups as lipase immobilization carriers were researched. Lipase immobilized in gigaporous microspheres was applied in synthesis of esters. The matching relations between pore size of carriers and molecular size of enzyme were initially investigated.There are mainly four sections in this dissertation.(1) The differences of mesoporous particles, gigaporous particles and a flat surface for enzyme immobilization were further investigated. The deeper reasons of the advantages of gigaporous microspheres were analyzed. By regulating the adsorption time and the enzyme concentration, it can be concluded that enzyme adsorbed in gigaporous microspheres can maintain better catalytic performance due to the low pore curvature, large pore space, the multilayer adsorption of enzyme and better mass transfer effects. When lipase adsorbed on a flat surface, the behavior was quite different from that in microspheres by excluding the influence of pores, the adsorption mass showed a linear increase and the specific activity had little change during the whole process. In addition, because of the accumulation of the substrate, the activity of enzyme could decrease with substrate of high concentration.(2) The gigaporous PGMA, P(ST-co-GMA), PST microspheres were used to be the carriers of lipase, which were compared with commercial immobilized lipase Novozym 435. Lipase immobilized in PGMA carriers showed the highest activity, reusability and stability. The influences of covalent binding and the hydrophobicity of carriers on the performance of immobilized lipase were investigated. Through a series of characterizations including the adsorption curve, the determination of density of epoxy groups, the contact angles measurement and the simple physical adsorption, it can be concluded that the advantages of PGMA microspheres were mainly due to the covalent binding between lipase and the carriers other than the relatively weaker hydrophobicity.(3) The synthesis of isooctyl palmitate catalyzed by lipase immobilized in gigaporous microspheres was investigated. Under the optimal reaction conditions, lipase in PGMA carriers can attain the highest esterification rate, which was 91.3%. The inactive immobilized lipase by reusing can be renatured by adding PBS. The activity of inactive PGMA-lipase can be renewed to over 80% of the initial activity.(4) Several kinds of enzymes with different molecular sizes were immobilized on meso- and giga-porous microspheres which were hydrophilic or hydrophobic. The relationship between molecular size of enzyme and pore size of carriers can be attained. The amylase with small molecular size (2~3 nm) can maintain relatively high activity in the microspheres with large pore size. The glucose oxidase (8.4 nm) was suitable to be adsorbed in hydrophilic carriers or immobilized in PGMA microspheres and the appropriate pore size was 100~200 nm. The catalase (10.4 nm) were suitable for the PGMA microspheres with pore size of 100~200 nm. Carriers with large pore size were beneficial to the enzyme immobilization. Covalent binding and protein loading can influence the activity retention of enzyme.In a word, gigaporous microspheres as enzyme immobilization carriers have great advantages due to the structure, the good mass transfer performance and the multilayer adsorption of enzyme. Lipase immobilization in gigaporous microspheres combined with covalent binding can improve its activity and stability. High catalytic efficiency, stability and reusability can be attained as lipase immobilized in gigaporous carriers. They were appropriate to immobilize various kinds of enzyme with different molecular sizes by regulating the microspheres, which can be ideal enzyme immobilization carriers.

语种中文
文献类型学位论文
条目标识符http://ir.ipe.ac.cn/handle/122111/22886
专题研究所(批量导入)
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王伟宸. 超大孔微球固定化酶体系的构建与应用[D]. 北京. 中国科学院研究生院,2016.
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