Activity and stability of enzymes immobilized in silica flow-through reactors via controllable adsorption of polymer-enzyme conjugates

Enzymes are biocatalysts that enable and regulate chemical transformations in living systems. Enzyme immobilization on solid supports aims to combine the advantages of these biocatalysts with the prospects of heterogeneous catalysis (easy product separation and catalyst reusability), including the use of flow-through reactor devices. In this thesis, polymer-enzyme conjugates were synthesized in aqueous solution and then adsorbed (immobilized) on silica surfaces in a controlled way for preparing enzymatic flow-through reactors. The enzymes used were horseradish peroxidase (HRP) and bovine carbonic anhydrase (BCA). For most parts of this work, a second generation dendronized polymer (denpol) was used. This polymer had on average around one thousand repeating units (de-PG21000), each containing four peripheral amino groups that rendered the polymer water-soluble at pH ≲ 8 and suitable for modification. Several copies of HRP or BCA were covalently bound to the same denpol chain using the UV/vis-quantifiable bis-aryl hydrazone (BAH) linker chemistry. After purification of the obtained conjugates, aqueous conjugate solutions were exposed to unmodified silica surfaces around pH 7 and thereby stably immobilized. The solid support used for most parts of this work was a cylindrical macro- and mesoporous silica monolith “MH1”. With the help of chromogenic substrates, the activity and stability of the immobilized enzymes were investigated in continuous flow-through reactions. The purified conjugate stock solutions were characterized in terms of properties that were crucial for a controlled enzyme immobilization. Once prepared, these stock solutions were stably storable and were suitable for reproducible flow-through reactor preparations over a prolonged period of time (more than one year with the chosen enzymes). While detailed investigations were carried out during optimization of the immobilization method, the elaborated procedure is simple and user-friendly. Simple exposure of defined volumes of conjugate solutions of defined enzyme concentrations to the porous silica surface of the monolith led to a stable and quantitative conjugate adsorption, as long as the internal surface was not saturated. This enabled full control over the amount of immobilized enzymes. When the activity of the immobilized enzymes was tested in flow-through assays, no active enzyme was found leaching from the monolith during operation. The flow-through activity of the reactors was found proportional to the amount of conjugate used during reactor preparation. The quantitative enzyme adsorption and the reproducible activity recovery upon immobilization allowed not only simple control over the conversion of enzyme substrates that were pumped through the monolith per time unit but also investigation of inherent properties of the immobilized enzymes. With the obtained methodology, the controlled co-immobilization of denpol-BCA and denpol-HRP conjugates was possible, in amounts and ratio as predetermined at will. A cascade reaction was investigated, which involved BCA-catalyzed hydrolysis, followed by HRP-catalyzed oxidation, using 2’,7’-dichlorodihydrofluorescein diacetate (DCFH2-DA) and hydrogen peroxide (H2O2) as initially added substrates. Before applying the cascade reaction to the immobilized enzymes, the reaction was investigated in detail in bulk solution with dissolved enzymes. Besides determining reference absorption spectra of all reaction components – allowing for an exact tracking of the reaction progression at any time –, it was discovered that the reaction can proceed along two different reaction pathways. Which pathway dominates in bulk solution is determined by the concentrations and ratio of BCA and HRP used. Comparative flow-through experiments with immobilized BCA and HRP showed that higher reaction yields in the case of co-immobilized enzymes as compared to sequentially immobilized enzymes originated from kinetic features of the cascade reaction itself and not from molecular proximity effects. In the final part of the thesis, a potential replacement of the denpol by commercially available α-poly-D-lysine (PDL) of similar length was assessed by using HRP as model enzyme. Neither during conjugate formation, conjugate adsorption nor resulting enzymatic flow-through reactor activity any advantage of the denpol over PDL was observed (using the porous silica monolith or glass micropipettes as solid support). With PDL-HRP conjugates immobilized inside the monolith, the quantification of H2O2 in diluted honey as a biological sample was possible in an efficient and reproducible way. Furthermore, stable flow-through operation at pH 6 – 7 was demonstrated, while the controlled desorption of the conjugate from the silica surface at pH 5 was shown, allowing the recovery of the support material. The presented immobilization method allows for a simple, versatile and efficient enzyme immobilization on unmodified silica surfaces for aqueous flow-through reactions, potentially for any type of enzyme. The method could be promising for demanding bioanalytical or small-scale synthetic applications that can profit from the controllability and versatility of the simple and reversible immobilization step, once long-term storable aqueous conjugate solutions are prepared

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

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

Enzyme-catalysed cascade reactions in flow-through systems with immobilised enzymes currently are of great interest for exploring their potential for biosynthetic and bioanalytical applications. Basic studies in this field often aim at understanding the stability of the immobilised enzymes and their catalytic performance, for example, in terms of yield of a desired reaction product, analyte detection limit, enzyme stability or reaction reproducibility. In the work presented, a cascade reaction involving the two enzymes bovine carbonic anhydrase (BCA) and horseradish peroxidase (HRP) – with hydrogen peroxide (H2O2) as HRP “activator” – was first investigated in great detail in bulk solution at pH = 7.2. The reaction studied is the hydrolysis and oxidation of 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA) to 2′,7′-dichlorofluorescein (DCF), which was found to proceed along two reaction pathways. This two-enzyme cascade reaction was then applied for analysing the performance of BCA and HRP immobilised in glass fiber filters which were placed inside a filter holder device through which a DCFH2-DA/H2O2 substrate solution was pumped. Comparison was made between (i) co-immobilised and (ii) sequentially immobilised enzymes (BCA first, HRP second). Significant differences for the two arrangements in terms of measured product yield (DCF) could be explained based on quantitative UV/vis absorption measurements carried out in bulk solution. We found that the lower DCF yield observed for sequentially immobilised enzymes originates from a change in one of the two possible reaction pathways due to enzyme separation, which was not the case for enzymes that were co-immobilised (or simultaneously present in the bulk solution experiments). The higher DCF yield observed for co-immobilised enzymes did not originate from a molecular proximity effect (no increased oxidation compared to sequential immobilisation)

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