Abstract
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.