Enzyme Shielding in a Large Mesoporous Hollow Silica Shell for Improved Recycling and Stability Based on CaCO3 Microtemplates and Biomimetic Silicification.

We report a novel "anchor-shield" approach for synthesizing a yolk-shell-structured biocatalytic system that consists of a phenylalanine ammonia lyase (PAL) protein particle core and a hollow silica shell with large mesopores by a combination of CaCO3 microtemplates and biomimetic silicification. The method is established upon filling porous CaCO3 cores with PAL via co-precipitation, controlled self-assembly and polycondensation of silanes, cross-link of the PAL molecules, and subsequent CaCO3 dissolution. During this process, the self-assembled layer of cetyltrimethylammonium bromide served as a structure-directing agent of the mesostructure and directed the overgrowth of the mesostructured silica on the external surface of PAL/CaCO3 hybrid microspheres; after CaCO3 dissolution, the cross-linked PAL particles were encapsulated in the hollow silica shell. The hollow silica shell around the enzyme particles provided a "shield" to protect from biological, thermal, and chemical degradation for the enzyme. As a result, the recycling of the PAL enzyme was improved remarkably in comparison to adsorbed PAL on CaCO3. PAL particles with a hollow silica shell still retained 60% of their original activity after 13 cycles, whereas adsorbed PAL on CaCO3 microparticles lost activity after 7 cycles. Moreover, immobilized PAL exhibited higher stability against a proteolytic agent, denaturants, heat, and extreme pH than adsorbed PAL on CaCO3 microparticles. These results demonstrated that the "anchor-shield" approach is an efficient method to obtain a stable and recycled biocatalyst with a yolk-shell structure.