Controllable Enzyme Immobilization via Simple and Quantitative Adsorption of Dendronized Polymer – Enzyme Conjugates Inside a Silica Monolith for Enzymatic Flow-Through Reactor Applications

Although many different methods are known for the immobilization of enzymes on solid supports for use in flow-through applications as enzyme reactors, the reproducible immobilization of predetermined amounts of catalytically active enzyme molecules remains challenging. This challenge was tackled using a macro- and mesoporous silica monolith as a support and dendronized polymer-enzyme conjugates. The conjugates were first prepared in an aqueous solution by covalently linking enzyme molecules and either horseradish peroxidase (HRP) or bovine carbonic anhydrase (BCA) along the chains of a water-soluble second-generation dendronized polymer using an established procedure. The obtained conjugates are stable biohybrid structures in which the linking unit between the dendronized polymer and each enzyme molecule is a bisaryl hydrazone (BAH) bond. Quantitative and reproducible enzyme immobilization inside the monolith is possible by simply adding a defined volume of a conjugate solution of a defined enzyme concentration to a dry monolith piece of the desired size. In that way, (i) the entire volume of the conjugate solution is taken up by the monolith piece due to capillary forces and (ii) all conjugates of the added conjugate solution remain stably adsorbed (immobilized) noncovalently without detectable leakage from the monolith piece. The observed flow-through activity of the resulting enzyme reactors was directly proportional to the amount of conjugate used for the reactor preparation. With conjugate solutions consisting of defined amounts of both types of conjugates, the controlled coimmobilization of the two enzymes, namely, BCA and HRP, was shown to be possible in a simple way. Different stability tests of the enzyme reactors were carried out. Finally, the enzyme reactors were applied to the catalysis of a two-enzyme cascade reaction in two types of enzymatic flow-through reactor systems with either coimmobilized or sequentially immobilized BCA and HRP. Depending on the composition of the substrate solution that was pumped through the two types of enzyme reactor systems, the coimmobilized enzymes performed significantly better than the sequentially immobilized ones. This difference, however, is not due to a molecular proximity effect with regard to the enzymes but rather originates from the kinetic features of the cascade reaction used. Overall, the method developed for the controllable and reproducible immobilization of enzymes in the macro- and mesoporous silica monolith offers many possibilities for systematic investigations of immobilized enzymes in enzymatic flow-through reactors, potentially for any type of enzyme.ISSN:2470-134

Performance of a Flow-Through Enzyme Reactor Prepared from a Silica Monolith and an α-Poly(D-Lysine)-Enzyme Conjugate

Horseradish peroxidase (HRP) is covalently bound in aqueous solution to polycationic α-poly(D-lysine) chains of ≈1000 repeating units length, PDL, via a bis-aryl hydrazone bond (BAH). Under the experimental conditions used, about 15 HRP molecules are bound along the PDL chain. The purified PDL-BAH-HRP conjugate is very stable when stored at micromolar HRP concentration in a pH 7.2 phosphate buffer solution at 4 °C. When a defined volume of such a conjugate solution of desired HRP concentration (i.e., HRP activity) is added to a macro- and mesoporous silica monolith with pore sizes of 20–30 µm as well as below 30 nm, quantitative and stable noncovalent conjugate immobilization is achieved. The HRP-containing monolith can be used as flow-through enzyme reactor for bioanalytical applications at neutral or slightly alkaline pH, as demonstrated for the determination of hydrogen peroxide in diluted honey. The conjugate can be detached from the monolith by simple enzyme reactor washing with an aqueous solution of pH 5.0, enabling reloading with fresh conjugate solution at pH 7.2. Compared to previously investigated polycationic dendronized polymer-enzyme conjugates with approximately the same average polymer chain length, the PDL-BAH-HRP conjugate appears to be equally suitable for HRP immobilization on silica surfaces.ISSN:1616-5187ISSN:1616-519

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

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

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

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