Stable Immobilization of Enzymes in a Macro- and Mesoporous Silica Monolith

Abstract

Horseradish peroxidase isoenzyme C (HRP) and Engyodontium album proteinase K (proK) were immobilized inside macro- and mesoporous silica monoliths. Stable immobilization was achieved through simple noncovalent adsorption of conjugates, which were prepared from a polycationic, water-soluble second generation dendronized polymer (denpol) and the enzymes. Conjugates prepared from three denpols with the same type of repeating unit (r.u.), but different average lengths were compared. It was shown that there is no obvious advantage of using denpols with very long chains. Excellent results were achieved with denpols having on average 750 or 1000 r.u. The enzyme-loaded monoliths were tested as flow reactors. Comparison was made with microscopy glass coverslips onto which the conjugates were immobilized and with glass micropipettes containing adsorbed conjugates. High enzyme loading was achieved using the monoliths. Monoliths containing immobilized denpol-HRP conjugates exhibited good operational stability at 25 °C (for at least several hours), and good storage stability at 4 °C (at least for weeks) was demonstrated. Such HRP-containing monoliths were applied as continuous flow reactors for the quantitative determination of hydrogen peroxide in aqueous solution between 1 μM (34 ng/mL) and 50 μM (1.7 μg/mL). Although many methods for immobilizing enzymes on silica surfaces exist, there are only a few approaches with porous silica materials for the development of flow reactors. The work presented is a promising contribution to this field of research toward bioanalytical and biosynthetic applications.