Flow‐through enzymatic reactors using polymer monoliths: From motivation to application

Abstract

The application of monolithic materials as carriers for enzymes has rapidly expanded to the realization of flow‐through analysis and bioconversion processes. This expansion is partly attributed to the absence from diffusion limitation in many monoliths‐based enzyme reactors. Particularly, the relatively ease of introducing functional groups renders polymer monoliths attractive as enzyme carriers. After summarizing the motivation to develop enzymatic reactors using polymer monoliths, this review reports the most recent applications of such reactors. Besides, the present review focuses on the crucial characteristics of polymer monoliths affecting the immobilization of enzymes and the processing parameters dictating the performance of the resulting enzymatic reactors. This review is intended to provide a guideline for designing and applying flow‐through enzymatic reactors using polymer monoliths.

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

“… 58 This effect of substrate depletion on the measured reaction rate is particularly relevant for surface-immobilized enzymes. 63 , 64 For immobilized enzymes that are assayed at a high substrate conversion, a high retention of the reaction product concentration in the outflow may not be due to a high stability of the immobilized enzymes. For operational stability data that directly reflect the true stability of the immobilized enzyme during substrate conversion, flow conditions must be applied that yield low substrate conversions (<20%).…”

“…When measuring single points on the reaction progression curve (as one is mostly forced to do in flow-through assays), using a too long reaction time significantly decreases the range within which a direct correlation exists between the measured amount of reaction product formed and the amount of the active enzyme. ,, As a consequence, at high substrate conversions, two similar measured product concentrations might originate from two very different concentrations of active enzyme; for an example, see Figure 1.5 in the work of Bisswanger . This effect of substrate depletion on the measured reaction rate is particularly relevant for surface-immobilized enzymes. , For immobilized enzymes that are assayed at a high substrate conversion, a high retention of the reaction product concentration in the outflow may not be due to a high stability of the immobilized enzymes. For operational stability data that directly reflect the true stability of the immobilized enzyme during substrate conversion, flow conditions must be applied that yield low substrate conversions (<20%).…”

Monolithic Papain-Immobilized Enzyme Reactors for Automated Structural Characterization of Monoclonal Antibodies

“…However, the higher K m , lower specific activity, and reduced k cat of papain immobilized on this material revealed that a lower percentage of enzyme was immobilized in its active form compared to the silica support. This result can be ascribed to the high hydrophobicity of polyHIPE material, which promotes enzyme adsorption yielding a larger amount of immobilized macromolecule, but also leads to enzyme unfolding with a consequent reduction of activity in most cases ( Mao et al, 2020 ). Although a direct comparison between kinetic parameters of free and immobilized enzymes is not possible due to the marked difference in terms of enzyme/substrate ratio and enzyme mobility, it was possible to observe a reduction in affinity to the substrate and activity per unit of enzyme after immobilization.…”

“…In particular, a K m increase of 50 (silica) or 100 (polyHIPE) folds, specific activity reduction of 100 (silica) or 150 (polyHIPE) folds, and k cat decrease of 40 (silica) or 60 (polyHIPE) folds were obtained from the IMERs compared to the free enzyme. This might be due to changes in the enzyme structure and/or inaccessibility of active sites as a result of the covalent immobilization, but also to long immobilization times which can have a negative impact on enzyme activity ( Mao et al, 2020 ). Nevertheless, the possibility to use IMERs for many digestion cycles allows to achieve greater yields and more reproducible reactions compared to free enzymes.…”

Monolithic flow reactor for enzymatic oxidations

“…In particular, porous monolith reactors are quickly emerging as an attractive path to continuous‐flow enzymatic reactions. When compared to other immobilization carriers, such as microbeads (packed bed reactors) 4 or membranes, 5 the biggest advantages of porous monoliths are their low to nonexistent pressure drop as well as large surface area for enzyme immobilization, which are both highly desired properties for continuous flow systems 6 . Other benefits include straightforward manufacturing and realistic scaling possibilities that are essential for future industrial applications.…”