新型模板法制备多孔脂肪酶微球及应用

To solve the low enzymatic activity, low loading capacity and the high cost of carrier in the preparation of carrier-bound enzyme, as well as the poor controlling of morphology and size of the particle in the preparation of carrier-free enzyme immobilization, porous carrier-free enzyme microspheres via template method were proposed in this project. Calcium carbonate was used as template while lipase was used as model enzyme. Firstly, the enzyme/calcium carbonate microsphere was prepared though the co-precipitation of enzyme, calcium chloride and sodium carbonate. Next, glutaraldehyde or dithiothreitol was added for cross-linking or assembling. After the template was removed, the porous lipase microsphere could be obtained. The effect of the immobilization conditions on porous lipase microsphere was studied, and the relationships between microenvironment of the enzyme and its catalytic properties were investigated, which was aimed to achieve the controllable preparation of the porous lipase microsphere. Finally, the enzyme microsphere was used to catalyze the preparation of glycerol carbonate. The effect of the reaction conditions on product yield was examined. The reaction process was also optimized for the efficient preparation of glycerol carbonate. The results of this research will provide novel theory and method for the design and preparation of novel carrier-free immobilized enzymes. Meanwhile, the results can also establish the foundation of the construction of other enzymes or mutienzyme system. Additionally, this research provides a new way to deal with glycerol which is the side product of biodiesel industry.

针对载体固定化酶制备过程中存在的单位质量酶活低、负载量低、载体成本高以及无载体固定化酶形貌、粒径、孔径可控性差等问题，提出通过模板法设计制备多孔无载体固定化酶微球的思路。本项目以碳酸钙为模板，脂肪酶为模型酶，首先通过酶分子、氯化钙和碳酸钠共沉淀制备酶/碳酸钙微球，接着加入戊二醛或二硫苏糖醇进行交联或组装，而后去除模板，即可制备得到多孔脂肪酶微球。考察固定化条件对多孔固定化酶微球的影响，探索酶所处微环境与催化特性间的关系，以实现高性能多孔脂肪酶微球的可控制备；最后将该固定化酶微球用于甘油碳酸酯的制备，考察反应条件对产物产率的影响，优化反应过程以实现甘油碳酸酯的高效制备。本项目研究成果将为新型无载体固定化酶微球设计与制备提供新的理论与方法，还可为其它酶以及多酶无载体固定化体系的构建奠定基础。另外，该研究为生物柴油生产过程中副产物甘油的处理提供了一条新出路。

为实现可控调节无载体固定化酶的形貌、粒径、孔径以及提高酶的负载量和酶活，本项目提出了以碳酸钙为模板，脂肪酶为模型酶，通过共沉淀法制备多孔脂肪酶微球的思路。利用酶分子、氯化钙和碳酸钠共沉淀制备了酶/碳酸钙微球，戊二醛交联或组装酶分子，去除碳酸钙模板，即得到了多孔脂肪酶微球。优化了固定化条件，探索了酶所处微环境与催化特性间的关系，研究了多孔脂肪酶微球的催化性能及稳定性。结果发现, 戊二醛浓度为100 mM，交联时间为2 h时，酶活回收率最高。相对于游离酶，多孔脂肪酶微球的pH和温度稳定性得到明显提高。对碳酸钙微球进行了表征，其比表面积为11.81 m2/g，孔径为27.75 nm，内部是由25-35 nm球霰石形成纤维状聚集体，由中心向外围发散。多孔脂肪酶微球的SEM表征结果外形为球形，表面粗糙，因此本项目实现了高性能多孔脂肪酶微球的可控制备；同时本项目还制备了介孔氧化硅纳米微球(D-MSNs)来固定化脂肪酶。以正硅酸乙酯为硅源，表面活性剂十六烷基三甲基氯化铵为模板，三乙醇胺为催化剂成功制备了D-MSNs，并对其进行了表征。结果显示D-MSNs直径约为177±14 nm，具有规则的孔道结构，比表面积为462 m2/g，孔体积为0.91 cm3/g，孔径为10.67 nm。将D-MSNs用于固定脂肪酶CALB得到固定化酶CALB@D-MSNs，并对其稳定性进行了测定。结果显示，CALB@D-MSNs拥有良好的水热稳定性、有机溶剂热稳定性以及有机溶剂耐受性。将其用于酯化反应中，重复使用10次后，油酸的转化率依然能达到85%左右，证明CALB@D-MSNs有良好的重复使用稳定性。最后，将固定化酶微球用于催化甘油和碳酸二甲酯反应以制备甘油碳酸酯，在优化的反应条件下甘油碳酸酯的产率为88.66%，甘油转化率为94.24%，实现了甘油碳酸酯的高效制备。本项目的研究成果为新型无载体固定化酶微球设计与制备提供了新的理论与方法，还为其它酶以及多酶固定化体系的构建奠定了基础。另外，该研究为生物柴油生产过程中副产物甘油的处理提供了一条新出路。