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黄曲霉毒素B1（AFB1）是一种对人和动物具有严重潜在危害的微量有机污染物，如何实现对其高效绿色去除是目前的研究热点。酶作为一种高效专一的生物催化剂已被广泛应用在微量有机污染物去除领域，但是由于其存在稳定性差、难以重复使用等缺点限制了其在工业上的应用。酶固定化有望解决以上问题，但同时也会降低酶的催化效率。针对以上问题，本论文重点研究了多功能酶固定化介质的制备，并分别利用介质的吸附和分离功能耦合固定化酶的催化功能，强化其对AFB1的去除。该研究所取得的成果主要包括以下三部分内容：受儿茶酚胺化学的启发，研究了单宁酸-3-氨基丙基三乙氧基硅烷（TA-APTES）涂层技术用于载体活化以及酶固定化的普适性。研究表明，与戊二醛、京尼平、聚多巴胺活化策略相比，TA-APTES涂层上由于具有多层次纳米结构和丰富的醌基导致其具有更优异的酶固定化性能。在TA-APTES涂层上二次接枝γ-聚谷氨酸后会进一步增加载酶量（3.5-4.5倍）。TA/APTES的比例和涂层时间均能影响酶的载量和比活力。当TA/APTES体积比为8:1和涂层时间为18 h时，涂层表现出最高的载酶量和比活性。TA-APTES涂层可活化各种基材（聚偏氟乙烯、聚醚砜、聚丙烯腈、尼龙膜、聚酯无纺布和不锈钢丝网），并通过共价键合固定不同种类的酶（辣根过氧化物酶（HRP）、脂肪酶、胃蛋白酶、漆酶和蔗糖酶），固定HRP的尼龙膜还可以用于多种微量有机污染物（双酚A、2,4,6-三氯苯酚、AFB1、呕吐毒素和四环素）的去除，证明了该涂层的普适性。AFB1易吸附在聚酰胺纳滤膜上并通过溶解扩散作用透过膜，导致其截留率不断下降。为了解决这一问题，我们将HRP共价固定在TA-APTES涂层修饰的NF270纳滤膜上，制备同时具有分离和催化功能的多功能膜。研究结果表明，该多功能膜由于涂层的阻隔效应（减少吸附）和缩孔效应（增加扩散阻力），使其对AFB1的截留率从75%上升至90%以上。H2O2的存在会启动酶促降解AFB1反应，进一步改善多功能膜对AFB1的去除效率。采用H2O2对污染后的多功能膜进行离线清洗后，其纯水通量可恢复至初始值的98.7%。为了探究固定化酶去除AFB1过程中可能存在的吸附-催化协同作用机制，分别制备了海藻酸/壳聚糖（SA/CS）微球和海藻酸/壳聚糖/蒙脱土（SA/CS/MON）杂化微球用于HRP的固定化。实验结果表明，与通过包埋和吸附交联的酶固定方法相比，通过共价键合法固定在微球表面的HRP具有更高的比活力和AFB1去除效率。荷负电的蒙脱土不仅能吸附大量的AFB1，还能在微球表面吸附更多的CS，为酶的固定化和交联提供更多的活性位点，从而使SA/CS/MON杂化微球具有更好的抗溶胀性能、更高的AFB1吸附容量（0.36 vs.4.5 μg/g）和载酶量（1.07 vs.1.25 mg/g）。尽管SA/CS/MON-HRP多功能微球对AFB1的强吸附能力并不能促进固定化酶的催化效率（三种可能的原因：底物抑制、产物吸附和降低主体溶液浓度），但其固定化酶的重复使用稳定性提高，其催化去除AFB1的能力随重复使用次数的增加而增加。这是因为随着微球的吸附饱和，主体溶液中的底物浓度会上升，从而提高了微球表面固定化酶的催化效率。由于微球中的吸附剂和表面的催化剂都可以设计甚至控制它们的空间分布，因此该多功能酶固定化介质在AFB1去除上具有极大的应用潜力。 ;As one of the toxic organic micropollutants, aflatoxin B1 (AFB1) is potentially detrimental to human beings and animals, and thus efficient and green removal of AFB1 is imperative. Enzyme, as a highly efficient and specific biocatalyst, has been widely used for the micropollutant removal. However, the industrial applications of enzymes are often hampered by several limitations such as poor stability and difficulty in reuse. Enzyme immobilization can possibly solve these problems, but it also lowers the catalysis efficiency of the enzyme. Aiming at these problems, this thesis focused on the preparation of multifunctional enzyme immobilization matrix, and the catalytic function of the immobilized enzyme was integrated with the adsorption or separation ability of the matrix to enhance the AFB1 removal. The main research contents and results were summarized as follows:Inspired by catecholamine chemistry, tannic acid-3-aminopropyltriethoxysilane (TA-APTES) coating was applied for carrier activation and subsequent enzyme immobilization, focusing on its universality on different carriers. It can be found that the TA-APTES coating showed much better performance in enzyme immobilization than glutaraldehyde, genipin, polydopamine activation strategies thanks to its special surface nanostructure and abundant quinone groups. Secondary grafting branched polymer γ-polyglutamic acid on the TA-APTES coating layer further increased enzyme loading (3.5-4.5 times). The TA/APTES ratio and coating time greatly affected enzyme loading and specific activity. The coating with a TA/APTES ratio of 8:1 and coating time of 18 h exhibited the highest enzyme loading and the best enzyme activity. Finally, the universality of TA/APTES coating was demonstrated by applying it on various materials (i.e. polyvinylidene fluoride, polyether sulfone, polyacrylonitrile, nylon membrane, polyester non-woven fabric and stainless steel wire mesh) for immobilizing different enzymes (i.e. horseradish peroxidase (HRP), lipase, pepsin, laccase and invertase) and removing five micropollutants (i.e. bisphenol A, 2,4,6-trichlorophenol, AFB1, deoxynivalenol and tetracycline).AFB1 easily adsorbed onto polyamide nanofiltration membrane, then dissolves into the membrane and passes through the membrane via diffusion, resulting in decreasing rejection with time. In order to solve this problem, HRP was covalently immobilized on NF270 nanofiltration membrane modified by TA-APTES coating to prepare multifunctional membrane with both separation and catalytic functions. The results showed that such a multifunctional membrane had a stable AFB1 rejection of more than 90% due to surface repulsion effect (reducing AFB1 adsorption) and pore narrowing effect (increasing diffusion resistance). The presence of H2O2 would induce the enzymatic reaction for AFB1 degradation, further improving the removal efficiency of AFB1 by the synergistic effect of the multifunctional membrane. After the fouled multifunctional membrane was cleaned by the dilute H2O2 solution, the water permeability can be restored to 98.7% of the initial value.In order to explore the possible adsorption-catalysis synergistic mechanism during the removal of AFB1 by the immobilized enzyme, alginate/chitosan (SA/CS) microspheres and alginate/chitosan/montmorillonite (SA/CS/MON) hybrid microspheres were prepared for HRP immobilization, respectively. Compared with the enzyme immobilization via encapsulation or adsorption followed by crosslinking, HRP immobilized on the microsphere surface via covalent bonding showed the highest specific activity and AFB1 removal efficiency. The negatively charged MON could not only adsorb abundant AFB1, but also attract more CS chains on the microsphere surface and bring more active sites for enzyme immobilization and crosslinking, thus the SA/CS/MON microspheres owned stronger anti-swelling ability, higher AFB1 adsorption capacity (0.36 vs.4.5 μg/g ) and enzyme loading (1.07 vs.1.25 mg/g). Although the AFB1 enrichment in the SA/CS/MON-HRP multifunctional microsphere did not promote its degradation by enzymatic catalysis (there are three possible reasons: substrate inhibition, product adsorption and lowing substrate concentration in bulk solution), the reuse stability of the multifunctional microsphere was improved and the enzymatic degradation efficiency of AFB1 increased with reuse cycle, which might be caused by increasing substrate concentration in the bulk solution when the adsorption capacity of the microsphere became saturate. The obtained multifunctional microsphere as a prototype is promising in the AFB1 removal because both the adsorbent and the catalyst can be designed, and their spatial distribution can even be controlled.
|周芳芳. 多功能酶固定化介质的制备及其在黄曲霉毒素B1去除上的应用[D]. 中国科学院大学,2020.|
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