【摘要】：固定化酶是酶研究和應(yīng)用中的重要內(nèi)容,多孔微球以其高比表面積、可控的親疏水性和功能基團(tuán)成為優(yōu)良的固定化酶載體,但常用的介孔微球會限制酶活性的發(fā)揮。本實驗室前期的研究證明,超大孔聚苯乙烯(PST)微球?qū)Ρ冉榭孜⑶蜃鳛楣潭ɑ久篙d體,在酶活、重復(fù)使用性、穩(wěn)定性以及動力學(xué)性質(zhì)方面更有優(yōu)勢。這些令人感興趣的結(jié)果促使本論文進(jìn)行深入研究,探索載體孔徑對固定化酶的影響機(jī)理,研究不同基質(zhì)的超大孔微球固定化酶的效果,并探討固定化酶的實際應(yīng)用,初步考察載體孔徑與酶分子大小的匹配關(guān)系。本論文主要分為四部分。(1)比較和研究脂肪酶在介孔微球、超大孔微球以及平面上的吸附狀態(tài),分析超大孔微球固定化酶具有優(yōu)勢的深層原因。通過對吸附時間和酶濃度的調(diào)控,闡明超大孔微球較低的孔曲率、較大的孔內(nèi)空間、吸附酶的多層狀態(tài)和更好的傳質(zhì)效果是其固定化酶比活和酶活較高的主要原因。脂肪酶吸附于平面時,由于沒有孔結(jié)構(gòu)的限制,酶活持續(xù)增長,比活基本保持不變。此外,底物濃度較高時,其富集作用會降低酶的催化效果。(2)比較不同基質(zhì)的超大孔聚甲基丙烯酸縮水甘油脂微球(PGMA)、苯乙烯-甲基丙烯酸縮水甘油酯共聚微球(P(ST-co-GMA))和PST微球作為脂肪酶固定化載體的效果,并與商品化固定化酶Novozym 435對比。結(jié)果表明,超大孔PGMA微球固定化酶有更高的酶活、更好的重復(fù)使用性、穩(wěn)定性和動力學(xué)性質(zhì)等�？疾旃矁r結(jié)合以及載體親疏水性對固定化酶性能的影響,通過吸附曲線、環(huán)氧基含量測定、接觸角測定以及單獨的物理吸附固定化酶等的考察,證明脂肪酶與載體的共價結(jié)合是PGMA微球具有顯著優(yōu)勢的主要原因。(3)考察超大孔微球固定化脂肪酶催化棕櫚酸異辛酯的合成反應(yīng),在最優(yōu)的反應(yīng)條件下,超大孔PGMA微球固定化酶催化反應(yīng)的酯化率可達(dá)91.3%,重復(fù)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緩沖液可以部分恢復(fù)活性,超大孔PGMA微球固定化酶可以恢復(fù)至初始活力的80%以上。(4)將不同分子大小的酶固定于不同孔徑、不同親疏水性的微球,得到載體選擇的規(guī)律,固定化酶載體的選擇很復(fù)雜,要綜合考慮孔徑、親疏水性、固定化方式以及底物傳質(zhì)等影響。大孔徑的載體可以很好保持酶活；根據(jù)酶和底物的性質(zhì)選擇不同親疏水性的載體；共價結(jié)合對于某些酶的活性和穩(wěn)定性有提升。同時也得到酶分子大小與載體孔徑間的匹配規(guī)律,酶分子尺寸與載體孔徑并不一定是正相關(guān)的關(guān)系,還要考慮載量的影響和底物和產(chǎn)物的傳質(zhì)要求。綜上所述,超大孔微球的結(jié)構(gòu)、優(yōu)良的傳質(zhì)性能和其中脂肪酶的多層吸附狀態(tài)使其作為固定化酶載體具有優(yōu)勢,與共價結(jié)合法聯(lián)合作用可以提高固定化脂肪酶的活性和穩(wěn)定性,在實際應(yīng)用中有較高的催化效率,通過對微球的調(diào)控,超大孔微球適用于多種類型酶的固定化,是一種理想的固定化酶載體。
[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.
【學(xué)位授予單位】：中國科學(xué)院研究生院(過程工程研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q814.2

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