“Fish‐in‐Net” Encapsulation of Enzymes in Macroporous Cages for Stable, Reusable, and Active Heterogeneous Biocatalysts

Abstract

There is currently great interest in enzyme immobilization to enhance enzyme stability and reusability, and to aid in separation from the reaction mixture, [1–17] but immobilized enzymes on commonly used inorganic and organic solid supports show low activities. This is a result of the leaching of the enzymes from the solid supports and the limited conformational transitions available to the enzymes for chemical interaction on the supports. [1–4] Enzymes encapsulated by a sol–gel/polymer [3–10] show good activity, but the wide pore-size distribution in sol–gel/polymers cannot be well controlled, and this adversely influences the diffusion of reactants and products during biocatalysis to the detriment of their practical application. [3,4,16] Recently, a number of successful examples of good enzyme activity resulting from enzyme immobilization in uniform mesopores of ordered mesostructured materials have been reported. [14–17] However, enzyme immobilization in mesopores is limited by the pore size of the mesostructured materials, so that bulky enzymes or enzyme aggregates larger than the mesopores cannot be immobilized. A general and facile approach for the encapsulation of enzymes of various sizes in ordered mesoporous silica is reported here, where the enzymes are entrapped in macroporous cages connected by uniform mesoporous channels. These encapsulated enzymes show good activity, long-term stability, and excellent recycling characteristics. The concept of “fish-in-net” encapsulation of enzymes in ordered mesoporous silica under mild conditions is illustrated in Figure 1. Tetraethylorthosilicate (TEOS) was first assembled from a triblock ethylene oxide (EO)/propylene oxide (PO) copolymer surfactant ( EO20PO70EO20, P123) in ethanol. After evaporation of the ethanol and addition of glycerol, preformed precursors with ordered mesostructured silica particles were obtained in the glycerol solution, which is a non-denaturing solvent for enzymes. The preformed precursors were mixed with the enzyme solution under stirring at 4°C. During the interaction between the enzymes and the preformed precursors, active enzymes (acting as the “fish”) were gradually entrapped in the “net” formed by the polymerization and condensation of the ordered mesostructured silica particles. After removal of the glycerol and water by evacuation, the xerogels with encapsulated enzymes were washed with ethanol and water several times to remove polymer surfactants in the mesopores. In contrast to the “ship-in-a-bottle” technique, [18] the enzymes in this work were used as templates for the formation of the macroporous cages. The encapsulated enzymes in the mesoporous silica are typical nanoreactors, which combine the advantages of native enzymes with those of mesoporous channels. When water is introduced into the cages, the chemical environment of the enzymes in the cages is similar to that of native enzymes in aqueous solution. This is beneficial for protein rotation and conformational transitions, and provides for high biocatalytic activity. [3,16] Moreover, the or