仿生固定化酶制备及其催化特性研究
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摘要
载体理性设计和制备是固定化酶领域的研究热点和前沿。本文借鉴酶在生物体中的存在形式,模仿细胞的多层结构,通过成分仿生、功能仿生和过程仿生设计和制备了核壳结构微囊,用于包埋β-D-葡萄糖醛酸苷酶(GUS),催化天然成分黄芩苷的高值转化。通过系统考察微囊液核、壳膜和杂化壳壁的结构和制备条件对GUS活力和稳定性的影响,尝试提出仿生固定化酶载体的制备原则。
首先,模仿细胞液的成分和功能选用三种具有不同电性的多糖(羧甲基纤维素钠、甲基纤维素和羧甲基壳聚糖钠)作为仿生微囊的液核,经比较可知,与酶分子具有静电排斥作用的多糖更有利于酶构象、活力的维持和酶稳定性的提高;然后,模仿细胞膜的结构和功能构建仿生微囊的壳膜。在适宜的制备条件下,多孔的海藻酸钙壳膜可在保证底物和产物自由扩散的同时,有效防止酶的泄漏;模仿硅藻细胞壁的形成过程和功能,首次利用精蛋白调控微囊表面的仿生硅化,形成规整的精蛋白/氧化硅杂化壳壁,完全抑制了微囊的溶胀,有效提高了微囊的重复使用稳定性。此外,还首次研究了精蛋白在仿生硅化过程中的催化作用和模板作用,并对仿生硅化的机理进行了初步探讨。
最终确定以羧甲基纤维素钠为仿生微囊的液核,制备出具有海藻酸/精蛋白壳膜和精蛋白/氧化硅杂化壳壁的微囊(APSi微囊),并用于包埋GUS,催化黄芩苷转化为黄芩素。其中羧甲基纤维素钠液核能够模仿GUS在生物体中的负电环境,提高GUS的活力和稳定性,尤其是在酸性条件下的pH稳定性和储存稳定性;海藻酸/精蛋白壳膜和精蛋白/氧化硅壳壁可实现GUS的高效包埋,防止GUS泄漏,同时提高了固定化GUS在碱性条件下的pH稳定性和重复使用稳定性。GUS在APSi微囊中的包埋率为69%,酶活力表现率为125%;在极端pH条件下,固定化GUS仍能维持85%以上的相对酶活力;储存26天后固定化GUS的相对酶活力仍高于90%;重复使用10次,固定化GUS的酶活力仍无明显下降;在37℃和pH7.0的条件下利用APSi微囊固定化的GUS水解黄芩苷,所得黄芩素的产率高达73%。
The key problem for enzyme immobilization is the rational design and preparation of carrier. In this study, a core-shell structured capsule was designed by mimicking the structure, component, function, and formation process of multi-layered cell.β-D-glucuronidase (GUS) encapsulated in this biomimetic capsule was utilized for the enzymatic conversion of baicalin to baicalein. The preparation conditions of capsule core, shell membrane and shell wall were systematically investigated, and their effects on the activity and stability of encapsulated GUS were extensively studied. Finally, the guideline for efficiently preparing core-shell structured capsule carrier was tentatively proposed.
The cationic, neutral and anionic polysaccharides were separately acted as the liquid core of biomimetic capsule by mimicking the component and function of cell sap. It was found that the electrostatic repulsion interaction between the polysaccharide core and the enzyme enhanced the enzyme activity and stability. The shell membrane of biomimetic capsule was then fabricated by mimicking the structure and function of cell membrane. Under the optimum preparation conditions, the porous Ca-alginate membrane of the capsule could not only confine the enzyme inside but also permit the substrate and product to freely diffuse in and out. Finally, mimicking the biosilicification process and function of diatom cell wall, protamine was for the first time utilized to mediate the formation of intact protamine/silica shell wall on the surface of capsule. The rigid silica shell wall dramatically inhibited the swelling of the capsule and significantly enhanced the recycling stability of capsule. In addition, the silica-precipitating and templating roles of protamine were elucidated, and the mediation mechanism of organic molecules in biomimetic silicification was also tentatively analyzed.
Baicalin was converted into baicalein by GUS encapsulated in biomimetic capsules. The sodium carboxymethyl cellulose (CMC) core created a biomimetic anionic microenvironment, and thus enhanced the activity of GUS. Meanwhile, the alginate/protamine shell membrane and the protamine/silica shell wall prevented the swelling of capsule and the leakage of GUS, and thus enhanced the recycling stability of GUS. The encapsulated GUS exhibited up to 125% of its free-form activity with an encapsulation efficiency of 69%. Moreover, the relative activity of encapsulated GUS under extreme pH conditions was up to 85%. The encapsulated GUS showed no appreciable loss in activity after 10 repeated cycles, and 90% of its initial activity remained after 26-day storage at 4 oC. Under the optimum conversion conditions (37℃, pH 7), a baicalein productivity as high as 73% was obtained.
首先,模仿细胞液的成分和功能选用三种具有不同电性的多糖(羧甲基纤维素钠、甲基纤维素和羧甲基壳聚糖钠)作为仿生微囊的液核,经比较可知,与酶分子具有静电排斥作用的多糖更有利于酶构象、活力的维持和酶稳定性的提高;然后,模仿细胞膜的结构和功能构建仿生微囊的壳膜。在适宜的制备条件下,多孔的海藻酸钙壳膜可在保证底物和产物自由扩散的同时,有效防止酶的泄漏;模仿硅藻细胞壁的形成过程和功能,首次利用精蛋白调控微囊表面的仿生硅化,形成规整的精蛋白/氧化硅杂化壳壁,完全抑制了微囊的溶胀,有效提高了微囊的重复使用稳定性。此外,还首次研究了精蛋白在仿生硅化过程中的催化作用和模板作用,并对仿生硅化的机理进行了初步探讨。
最终确定以羧甲基纤维素钠为仿生微囊的液核,制备出具有海藻酸/精蛋白壳膜和精蛋白/氧化硅杂化壳壁的微囊(APSi微囊),并用于包埋GUS,催化黄芩苷转化为黄芩素。其中羧甲基纤维素钠液核能够模仿GUS在生物体中的负电环境,提高GUS的活力和稳定性,尤其是在酸性条件下的pH稳定性和储存稳定性;海藻酸/精蛋白壳膜和精蛋白/氧化硅壳壁可实现GUS的高效包埋,防止GUS泄漏,同时提高了固定化GUS在碱性条件下的pH稳定性和重复使用稳定性。GUS在APSi微囊中的包埋率为69%,酶活力表现率为125%;在极端pH条件下,固定化GUS仍能维持85%以上的相对酶活力;储存26天后固定化GUS的相对酶活力仍高于90%;重复使用10次,固定化GUS的酶活力仍无明显下降;在37℃和pH7.0的条件下利用APSi微囊固定化的GUS水解黄芩苷,所得黄芩素的产率高达73%。
The key problem for enzyme immobilization is the rational design and preparation of carrier. In this study, a core-shell structured capsule was designed by mimicking the structure, component, function, and formation process of multi-layered cell.β-D-glucuronidase (GUS) encapsulated in this biomimetic capsule was utilized for the enzymatic conversion of baicalin to baicalein. The preparation conditions of capsule core, shell membrane and shell wall were systematically investigated, and their effects on the activity and stability of encapsulated GUS were extensively studied. Finally, the guideline for efficiently preparing core-shell structured capsule carrier was tentatively proposed.
The cationic, neutral and anionic polysaccharides were separately acted as the liquid core of biomimetic capsule by mimicking the component and function of cell sap. It was found that the electrostatic repulsion interaction between the polysaccharide core and the enzyme enhanced the enzyme activity and stability. The shell membrane of biomimetic capsule was then fabricated by mimicking the structure and function of cell membrane. Under the optimum preparation conditions, the porous Ca-alginate membrane of the capsule could not only confine the enzyme inside but also permit the substrate and product to freely diffuse in and out. Finally, mimicking the biosilicification process and function of diatom cell wall, protamine was for the first time utilized to mediate the formation of intact protamine/silica shell wall on the surface of capsule. The rigid silica shell wall dramatically inhibited the swelling of the capsule and significantly enhanced the recycling stability of capsule. In addition, the silica-precipitating and templating roles of protamine were elucidated, and the mediation mechanism of organic molecules in biomimetic silicification was also tentatively analyzed.
Baicalin was converted into baicalein by GUS encapsulated in biomimetic capsules. The sodium carboxymethyl cellulose (CMC) core created a biomimetic anionic microenvironment, and thus enhanced the activity of GUS. Meanwhile, the alginate/protamine shell membrane and the protamine/silica shell wall prevented the swelling of capsule and the leakage of GUS, and thus enhanced the recycling stability of GUS. The encapsulated GUS exhibited up to 125% of its free-form activity with an encapsulation efficiency of 69%. Moreover, the relative activity of encapsulated GUS under extreme pH conditions was up to 85%. The encapsulated GUS showed no appreciable loss in activity after 10 repeated cycles, and 90% of its initial activity remained after 26-day storage at 4 oC. Under the optimum conversion conditions (37℃, pH 7), a baicalein productivity as high as 73% was obtained.
引文
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