The development of process steps catalyzed by immobilized enzymes usually encompasses the screening of enzyme variants, as well as the optimization of immobilization protocols and process parameters. Direct immobilization of biocatalysts by physical entrapment into hydrogels can be applied to reduce the effort required for immobilization, as the enzyme-specific optimization of the immobilization procedure is omitted. Physical entrapment is applicable for purified enzymes as well as crude cell extracts. Therefore, it can be used to quickly assess and compare activities of immobilized enzymes. For the application in flow reactors, we developed 3D-printed hydrogel lattices for enzyme entrapment as well as matching housings, also manufactured by 3D-printing. Testing the resulting enzyme reactors for three different enzymes, namely alcohol dehydrogenase from Lactobacillus brevis, benzoylformate decarboxylase from Pseudomonas putida and β-galactosidase from Aspergillus oryzae, and four different enzymatic reactions showed the broad applicability of the approach but also its limitations. The activity of the immobilized biocatalysts was measured in batch experiments and compared to the kinetics of the respective free enzymes in solution. This comparison yields an effectiveness factor, which is a key figure to describe the extent the immobilized catalyst is effectively utilized. For the examined systems the effectiveness factor ranged between 6 and 14% and decreased with increasing absolute activity of the entrapped enzymes due to mass transfer limitations. To test the suitability of the hydrogel lattices for continuous operation, they were inserted into 3D-printed reactor housings and operated at constant flow. Stable product formation could be monitored over a period of 72 h for all four enzymatic systems, including two reactions with redox cofactor regeneration. Comparing calculated and experimental conversion in the continuous setup, higher values of the effectiveness factor in batch experiments also hint at good performance in continuous flow. This can be used to optimize complex biocatalytic reactions on a small scale.

Physical entrapment of enzymes within a porous matrix is a fast and gentle process to immobilize biocatalysts to enable their recycling and long‐term use. This study introduces the development of a biocompatible 3D‐printing material suitable for enzyme entrapment, while having good rheological and UV‐hardening properties. Three different viscosity‐enhancing additives have been tested in combination with a poly(ethylene glycol) diacrylate‐based hydrogel system. The addition of polyxanthan or hectorite clay particles results in hydrogels that degrade over hours or days, releasing entrapped compounds. In contrast, the addition of nanometer‐sized silicate particles ensures processability while preventing disintegration of the hydrogel. Lattice structures with a total height of 6 mm consisting of 40 layers were 3D‐printed with all materials and characterized by image analysis. Rheological measurements identified a shear stress window of 200 < τ < 500 Pa at shear rates of 25 s−1 and 25°C for well‐defined geometries with an extrusion‐based printhead. Enzymes immobilized in these long‐term stable hydrogel structures retained an effective activity of approximately 10% compared to the free enzyme in solution. It could be shown that the reduction of effective activity is not caused by a significant reduction of the intrinsic enzyme activity but by mass transfer limitations within the printed hydrogel structures.

No abstract

We propose surface acoustic wave (SAW) resonators as a complementary tool for conditioning film monitoring. Conditioning films are formed by adsorption of inorganic and organic substances on a substrate the moment this substrate comes into contact with a liquid phase. In the case of implant insertion, for instance, initial protein adsorption is required to start wound healing, but it will also trigger immune reactions leading to inflammatory responses. The control of the initial protein adsorption would allow to promote the healing process and to suppress adverse immune reactions. Methods to investigate these adsorption processes are available, but it remains difficult to translate measurement results into actual protein binding events. Biosensor transducers allow user-friendly investigation of protein adsorption on different surfaces. The combination of several transduction principles leads to complementary results, allowing a more comprehensive characterization of the adsorbing layer. We introduce SAW resonators as a novel complementary tool for time-resolved conditioning film monitoring. SAW resonators were coated with polymers. The adsorption of the plasma proteins human serum albumin (HSA) and fibrinogen onto the polymer-coated surfaces were monitored. Frequency results were compared with quartz crystal microbalance (QCM) sensor measurements, which confirmed the suitability of the SAW resonators for this application.