Among enzyme immobilization techniques, the preparation of cross-linked enzyme aggregates has shown promising results in biocatalysis, because they are easy to prepare, versatile, and cheap. The method involves the precipitation of enzymes with ammonium sulfate or an organic solvent and subsequent cross-linking with glutaraldehyde. However, the Schiff base produced with glutaraldehyde is reversible and can be broken with acids or bases, releasing proteins to the reaction medium. To solve this problem, we propose replacing glutaraldehyde with diepoxide compounds to obtain an irreversible secondary amine bond. Such a substitution avoids protein leakage during the biocatalytic process, contamination of the final products, and loss of enzyme. It also improves the synthesis of the biocatalyst, because, while the Schiff base is favored at mildly acidic pH, the epoxide reaction can be made at the optimal enzyme pH, assuring its structural stability and catalytic performance. The proposed method has been successfully used in the production and optimization of aldolase epoxy-cross-linked aggregates, which retain 98% activity. (C) 2017 American Institute of Chemical Engineers