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Ficin extract has been immobilized on different 4% aminated-agarose beads. Using just ion exchange, immobilization yield was poor and expressed activity did not surpass 10% of the offered enzyme, with no significant effects on enzyme stability. The treatment with glutaraldehyde of this ionically exchanged enzyme produced an almost full enzyme inactivation. Using aminated supports activated with glutaraldehyde, immobilization was optimal at pH 7 (at pH 5 immobilization yield was 80%, while at pH 9, the immobilized enzyme became inactivated). At pH 7, full immobilization was accomplished maintaining 40% activity versus a small synthetic substrate and 30% versus casein. Ficin stabilization upon immobilization could be observed but it depended on the inactivation pH and the substrate employed, suggesting the complexity of the mechanism of inactivation of the immobilized enzyme. The maximum enzyme loading on the support was determined to be around 70 mg/g. The loading has no significant effect on the enzyme stability or enzyme activity using the synthetic substrate but it had a significant effect on the activity using casein; the biocatalysts activity greatly decreased using more than 30 mg/g, suggesting that the near presence of other immobilized enzyme molecules may generate some steric hindrances for the casein hydrolysis.

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.

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