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.

A two-enzyme cascade reaction consisting of two reaction pathways. Studies in bulk solution for understanding the performance of a flow-through device with immobilised enzymes

“…For the BCA-conjugates, details about their synthesis, puri-cation and quantication are given in (Yoshimoto et al, 2018); 52 for the HRP-conjugate, see (Hou et al, 2019). 59 Additional information is provided in the ESI (Section 4) with 2.5. Immobilisation of de-PG2 1000 -BAH-BCA and de-PG2 1000 -BAH-HRP in glass ber lters…”

“…52 The corresponding concentration of BCA in this desalted denpol-BCA conjugate solution was [BCA] ¼ 8.8 mM. The denpol-HRP conjugate solution used was prepared from a stock solution obtained from the conjugate preparation according to a previously published protocol using 59 For control measurements with "empty" lters, pure PB or pure 10 mM sodium phosphate buffer solution (pH ¼ 7.0, 0.15 M NaCl) was used.…”

“…The two enzymes BCA and HRP were immobilised in borosilicate glass ber lters with a methodology that has been elaborated before, see (Yoshimoto et al, 2018; Hou et al, 2019). 52,59 The method is based on rst preparing and characterising covalent dendronised polymer (denpol)-enzyme conjugates in bulk aqueous solution. Dened volumes of aqueous solutions of such conjugates at dened enzyme concentrations were then added to the glass ber lters and incubated for 1 h at room temperature.…”

“…The dendronised polymer used for the preparation of the conjugates was the same as in our previous work, 52,59 abbreviated as de-PG2 1000 (Fig. 1).…”

“…S-4and S-5. † If stored in aqueous solution at 4 C, both types of conjugates are stable over prolonged periods of time 52,59.…”