Advantages of Hydrogel-Based 3D-Printed Enzyme Reactors and Their Limitations for Biocatalysis

Abstract

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.

An Enzymatic 2‐Step Cofactor and Co‐Product Recycling Cascade towards a Chiral 1,2‐Diol. Part II: Catalytically Active Inclusion Bodies

“…As demonstrated in Figure 2 the reaction with the soluble enzymes initially proceeded faster until a plateau was reached after 2 days, after which no significant further formation of PPD was observed (Figure 2 B,C). The somewhat slower conversion observed for both CatIBs and Co-CatIBs might be related to diffusional/mass transfer limitation which is known for enzyme immobilizates in general [12] and for CatIBs in particular. [11f] In all cases tested with this intial setup significant amounts of the cascade intermediate HPP accumulated during the course of the reaction ( Figure 2, A and C; dashed lines; Table 1, entries 1-6).…”

Enzyme Scaffolds with Hierarchically Defined Properties via 3D Jet Writing

“…The reactor was 3D-printed with VeroWhitePlus RGD835 (Stratasys, Eden Prairie, MN) as described in other publications. [36,43] Due to adsorption effects, [36] the reactor was rinsed with oNP solution for several hours to guarantee a completely saturated reactor wall. Before the start of the experiment, the reactor was rinsed with citrate buffer until oNP was no longer detectable in the outlet.…”

“…This is around twice as high as comparable experiments with 3D-printed scaffolds. [36] The fluctuations of the recorded results are due to bleaching of the product solution during overnight hours. At the end of the experiments with a duration of 76 h, all scaffolds were still intact without any noticeable damage.…”

Miniaturization and 3D Printing of Bioreactors: A Technological Mini Review

“…This shows that 3D printing can be applied to many different applications in many different sizes ( Table 3 ). The conditions also range widely between 37 °C for immobilizing enzymes in hydrogel lattices [ 86 ] and an injector temperature of 200 °C for gas chromatography [ 85 ]. Two of the found researches are about bioreactors; one of them is used for mechanical stretching and tissue engineering.…”