Activity and stability of enzymes immobilized in silica flow-through reactors via controllable adsorption of polymer-enzyme conjugates
Author / Producer
Date
2022
Publication Type
Doctoral Thesis
ETH Bibliography
yes
Citations
Altmetric
Data
Rights / License
Abstract
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.
Permanent link
Publication status
published
External links
Editor
Contributors
Examiner: Walde, Peter
Examiner: Niederberger, Markus
Examiner: Dufresne, Eric
Examiner : Shahgaldian, Patrick
Book title
Journal / series
Volume
Pages / Article No.
Publisher
ETH Zurich
Event
Edition / version
Methods
Software
Geographic location
Date collected
Date created
Subject
Enzyme immobilization; Flow-through reactor; ENZYMATIC ACTIVITY (BIOCHEMISTRY); Biocatalysis; ENZYME KINETICS (BIOCHEMISTRY); Enzymes; POLYLYSINE (PROTEINS AND PEPTIDES); Dendronized polymer; UV/Vis spectroscopy
Organisational unit
03763 - Niederberger, Markus / Niederberger, Markus