Efficient retention of laccase by non-covalent immobilization on amino-functionalized ordered mesoporous silica

Abstract The present work aims to be a step forward in the synthesis of siliceous ordered mesoporous materials (OMM) as tailor made matrices to optimize the immobilization and stabilization of enzymes. Based on a classic non-covalent adsorption by electrostatic interactions we have developed the syntheses of materials especially designed for this enzyme, in order to optimize the properties of the final biocatalyst. Siliceous materials with a hexagonal arrangement of parallel mesoporous channels (SBA-15 type of structure) have been synthesized, whose pore diameter has been tuned according to the molecular dimensions of laccase. The synthesis conditions used allowed to obtain pore sizes large enough to permit laccase entrance and diffusion through the pore channels. Diffusion of the enzyme is crucial to obtain high immobilization yield since most of the surface area of the particles is the internal surface of the pores. A poor diffusion would involve retention of enzyme molecules in the pore mouths preventing new ones to access the channel and leading to a low enzyme loading of the catalyst. A micelle swelling agent has been used to expand the supramolecular aggregates that generate the pore architecture of SBA-15 silica. The surfaces of the supports were functionalized with amino groups aiming to strengthen electrostatic interactions between support and enzyme at a suitable pH. Two strategies of surface functionalization of the large-pore ordered mesoporous silica materials were followed: (1) anchoring of an amino-functional alkoxysilane on mesoporous silica and (2) direct co-condensation of a silicon alkoxide and an amino-functional alkoxysilane to obtain the functionalized material in one step. The possibility to prepare carriers where each characteristic has been separately studied and optimized has allowed to obtain biocatalysts with optimal properties. Enzyme loading up to 187 mg/g of catalyst and high activities were achieved with the amino-functionalized large-pore supports. Furthermore, immobilization improved enzyme stability in ethanol. Strong binding forces were capable of housing and retaining the enzyme irreversibly, fully preventing leaching in aqueous medium. Through the careful design of the support material, the biocatalysts obtained share the advantages of enzyme-support covalent attachment regarding absence of leaching and stability, while avoiding drawbacks like loss of activity and enabling the reuse of the support.