Vinyl sulfone (VS)–agarose beads were used to develop a new strategy to coimmobilize enzymes with very different stabilities, enabling the reuse of the most stable immobilized enzyme. Two model combi-biocatalysts were prepared. First, trypsin and chymotrypsin were multipoint covalently coimmobilized on VS–agarose, the support was blocked with ethylenediamine, and then β-galactosidase from Aspergillus oryzae was coimmobilized via anion exchange. Both immobilized proteases were more stable than the immobilized lactase, which could be released from the triple combi-biocatalyst by incubation in 400 mM ammonium sulfate at pH 7.0. Four cycles of combi-biocatalyst incubation at high temperature, β-galactosidase partial inactivation, and release and reimmobilization of the β-galactosidase in the combi-protease biocatalysts were performed, maintaining 80% of the activity of immobilized trypsin and 90% of immobilized chymotrypsin. The second model system was the coimmobilization of trypsin and ficin. Ficin was inactivated when immobilized on VS–agarose supports, but it could be immobilized on this support blocked with aspartic acid. Trypsin was covalently immobilized on the support blocked with aspartic acid and ficin was coimmobilized via cation exchange. As the covalently immobilized enzyme was more stable than ionically exchanged ficin, the combi-biocatalyst was submitted to four cycles as described above with similar results.

Alcalase was scarcely immobilized on monoaminoethyl‐N‐aminoethyl (MANAE)‐agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE‐agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc‐Ala‐ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.