酶固定化的杂化微凝胶的可控制备及其吸附与降解染料研究

Development of novel carriers to create a suitable microenvironment for the immobilized enzyme and manipulation the interaction between the enzyme and carriers are crucial for the application of immobilized enzyme in the decolorization and degradation of dyes from wastewater. The composition, morphology, and surface characteristics of carriers play critical roles in regulating enzyme activity. The object of this project is to design, preparation and characterization of a novel alginate/graphene oxide (Alg-GO) hybride microgel with controlled structure and size. The Alg-GO hybrid microgel with a large number of absorbed and entrapped enzymes will be fabricated through horseradish peroxidase (HRP)-catalyzed hydrogelation using a microfluidic system. The HRP or laccase will be absorbed on GO surface and encapsulated in the network of microgel simultaneously during the process of hydrogelation. The enzyme-friendly HRP-catalyzed hydrogelation allows enzymes to be incorporated in the microgel without compromising their enzymatic activity. The interaction between GO, enzyme and alginate, the mechanism of the stabilization of gel precursor will be discussed in detail. The suitable microenvironment for immobilized HRP or laccase will be create by tailoring the cross-link density, mesh size and the interaction between enzyme and carrier. The influence of the structure and property Alg-GO hybride microgel on the amount, specific activity, thermal and storage stability, efficiency of enzymatic degradation of dyes of immobilized HRP or laccase will be studied in detail. The HRP or lacasse immobilized Alg-GO hybrid microgel will be applied in the absorption and enzymatic degradation of dyes from wastewater. We believe that the novel hydrid microgel with immobilized HRP or laccase will effectively absorb and degrade dyes. The efficiency of enzymatic degradation of dyes by immobilized HRP or laccase will be examined and the optimal processing parameters of the enzymatic degradation of dyes should be obtained. The purpose of this project is to provide an innovative idea for designing and developing of enzyme immobilized microgel to improve efficiency of enzymatic degradation of dyes. This project will generate key experimental basis and research samples of immobilized enzymes for further industry application in the treatment of dyes wastewater.

选择合适的载体构建适宜于酶催化反应的微环境和调控酶与载体材料的相互作用是实现固定化酶对染料高效脱色与降解的关键，本项目设计将辣根过氧化物酶（HRP）催化的酶促凝胶化反应与微流体技术相结合可控制备原位包埋氧化石墨烯（GO）和酶（HRP或漆酶）的海藻酸杂化微凝胶，利用GO对酶的高效吸附和水凝胶对酶的无损害包埋获得高活性的固定化酶体系，通过调控杂化微凝胶的网孔尺寸、交联密度、酶与载体的相互作用构建出适宜酶催化降解染料的微环境，系统研究杂化微凝胶结构和性能对酶固载量、固定化酶的活性、稳定性和催化降解染料效率的影响，建立杂化微凝胶结构与性能的经验关系。利用杂化微凝胶对染料的高容量吸附和酶催化的高效降解有效地提高染料废水处理效果，获得固定化酶处理染料废水的适宜工艺条件。本项目的实施有望获得一类具有实用价值的染料废水处理用固定化酶体系，为酶固定化载体的设计和杂化微凝胶的可控制备提供创新思路和关键技术。

为实现固定化酶对染料高效脱色与降解，本项目利用酶促凝胶化反应开发几种杂化水凝胶和纳米凝胶固定化酶。结合离子交联法和酶催化交联反应制备了双交联的氧化石墨烯/海藻酸钠杂化水凝胶固定化酶。该固定化酶明显地改善了HRP的热稳定性和酸碱适应性，在游离酶几乎完全失活的极端条件下（pH 2或70 ℃的情况下），固定化酶仍可保持有40 %左右的脱色率。固定化酶对染料对染料存在吸附与降解双重处理作用，动力学曲线符合二级动力学模型，可应用于染料废水的高效脱色与降解，在150 min内染料的脱色率高达82 %，经过12个循环使用周期后脱色率仅仅下降了17 %。结合两亲性嵌段共聚物在水中的胶束化和酶催化交联技术制备了固定化酶的纳米凝胶，并实现了对纳米凝胶固定化酶的活性调控。开发了一种综合性能优良的聚丙烯酸/GO纳米复合水凝胶，应用于染料废水中阳离子染料的高效吸附。复合水凝胶对亚甲基蓝MB的吸附表现出明显的pH敏感性，在pH 3~11的溶液中，MB的吸附量（qe）增大变化量高达1400～1500 mg/g，pH 11时最大的qe高达1789 mg/g。复合水凝胶具有良好的重复使用性，三次吸附-解吸附循环后对MB的吸附能力仍高于70 %，解吸附效率高于90%。利用酶促交联反应合成了两种近红外光热响应（NIR）的GO/温敏聚合物纳米复合水凝胶，成功应用于NIR响应的药物控制释放。通过酶催化交联合成贻贝仿生的可注射水凝胶，可通过改变温度调控其粘附性。结合LBL与酶催化交联制备了二硫键交联的还原响应性微胶囊，实现了细胞内蛋白质输送与还原响应释放。本项目的研究拓展了酶促交联反应的应用领域，获得了几种具有实用价值的染料废水处理用环境保护材料，为酶固定化载体的设计和功能性可注射水凝胶的可控制备提供了创新思路和关键技术。