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。

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.