生物酶接枝聚合物及其在微通道反应器的固定机理研究

Under the impetus of green chemistry and sustainable development, simple, the efficient, and long-lasting enzyme immobilization methods have broad application prospects in the fields of industry and agriculture. The traditional enzyme immobilization methods only emphasize the retention of its activity, and these methods have the disadvantages of complex procedure, limited repeated catalysis cycles, discontinuous operation and difficult industrialization. In this project, a graft polymer cross-linking enzyme was used to immobilize enzyme in a silicon oxide microchannel reactor, which is in a glass tube, and the enzyme was immobilized by adsorbing a biopolymer-grafted polymer from an aqueous solution. Firstly, this conjugate was first synthesized from a polycationic dendronized polymer and enzyme. The enzyme were attached along the denpol chain via stable bis-aryl hydrazone bonds. There are 5-15 enzyme molecules on an average of 100 repeating units of dendronized polymer. Secondly, the purpose of controlling the catalytic activity of the bio-enzyme grafted polymer is achieved by effectively regulating the structure and composition of the bio-enzyme grafted polymer. Finally, the immobilized behavior of immobilized polymer-enzyme in microchannel reactor, the immobilized catalytic activity, the possibility of catalytic chain reaction, and the immobilization mechanism of biological enzyme were studied. In this study, the enzyme molecules were immobilized in microchannel flow reactor by cross-linking, adsorption, embedding and covalent bonding. This work will break through the traditional preparation idea of immobilized enzyme, and enhance the application value and industrial prospect of immobilized enzyme. This work will also provide a more scientific basis for the further development of immobilized enzymes.

在绿色化学和可持续发展的推动下，简单、高效、持久的酶固定化方法在工农业领域有广阔的应用前景。传统酶固定化方法只强调保留其活性，方法复杂，重复使用次数有限，难以连续操作，工业化困难。本项目采用接枝聚合物交联酶，将氧化硅微通道反应器固定于玻璃管中，通过从水溶液吸附生物酶接枝聚合物实现酶的固定化。首先，该生物酶接枝聚合物由聚阳离子树枝状聚合物和生物酶通过稳定的双芳基腙键共价连接，控制平均100个重复单元上有5-15个酶分子。其次，通过有效调节生物酶接枝聚合物的结构和组成，达到控制生物酶接枝聚合物催化反应活性的目的。最后，研究生物酶接枝聚合物在微通道反应器的固定行为、固定后的催化活性、催化连锁反应的可能性、与生物酶的固定机理。本项目中酶分子通过交联、吸附、包埋、共价结合等复合方法固定于微通道反应器中，将突破固定化酶传统制备思路，提升固定化酶的应用价值和工业前景，为固定化酶的进一步发展提供理论依据。

本项目首先以二代阳离子型的聚甲基丙烯酸类接枝聚合物(PG2)为主链，经醛肼反应得到生物酶接枝聚合物(de-PG2-BAH-HRP，de-PG2-BAH-proK 和 de-PG2-BAH-(HRP，proK))，研究其在玻璃片表面、玻璃管内部、微通道反应器中的固定性能和催化活性稳定性；其次，基于羧甲基纤维素（CMC），以甲基甲基丙烯酰氧乙基三甲基氯化铵（DMC）为功能单体，过硫酸钾（KPS）为引发剂引发二者接枝共聚，经戊二醛（GA）改性后，得到阳离子型聚羧甲基纤维素CMC-g-(PDMC-r-GA)；经GA上醛基和辣根过氧化物酶（HRP）反应，HRP和聚合物CMC-g-(PDMC-r-GA)进行接枝共聚，得到CMC-g-(PDMC-r-GA)/HRP；通过吸附法交联法将生物酶接枝聚合物分别固定在盖玻片和玻璃微管上。通过FTIR、XRD、TG分别对聚合物的结构和性能进行了表征。并用紫外可见分光光度计（UV/vis）对固定化酶的活性以及稳定性进行了表征。结果表明，CMC-g-(PDMC-r-GA)的成功制备，与HRP接枝固定后，生物酶保持良好的稳定性和活性，在A414nm处的吸光度达到0.15左右。最后，基于羧甲基纤维素（CMC），以甲基丙烯酸缩水甘油酯（GMA）为功能单体，过硫酸钾引发二者接枝共聚，经乙二胺（EDA）和戊二醛改性后，得到阳离子型聚合物CMC-g-( PGMA/NH2-r-GA)；再经与酶交联后，得到CMC-g-( PGMA/NH2-r-GA)/HRP；通过吸附交联法将酶固定在玻璃载体商，并用紫外分光光度计（UV/vis）对酶的活性和稳定性进行测量。结果表明，固定后的生物酶活性较高，在414nm处的吸光度高达0.8左右。