A cascade reaction is defined as the chemical process which consists of two or more consecutive reactions carried out in the same reaction vessel due to the multifunctionality of the catalyst. This approach was utilized by researchers to design advanced biocatalysts with two or more enzymes immobilized on the same support. The key benefits of multienzyme biocatalysts rise from a single pot, where consecutive reactions are performed, thus eradicating the handling of intermediate products. This makes processes more sustainable, allowing one to obtain target molecules in a more environmentally friendly way. We report on the effect of pore size in biocatalysts based on mesoporous silica on the loading of immobilized enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP) as well as on the activity and the possibility of reuse of the bio-enzyme biocatalyst in a cascade reaction. The choice of silica substrates was due to their commercial availability and sharply different pore sizes: 6 nm and 15 nm. In the first case, the pores are too small to accommodate GOx and HRP, the hydrodynamic diameter of which is ~ 8 nm for each, while in the second case, there is enough space for immobilization of GOx and HRP inside the pores. We demonstrate that larger pores provide higher enzyme throughput due to immobilization within the pores, remarkable 95% relative activity, and increased stability during reuse due to the conformational integrity of GOx and HRP in spacious pores. This study clearly shows the advantages of substrates with pores larger than the size of the enzyme for its immobilization during the synthesis of the biocatalyst.
