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

Barbara Schmieg,Johannes Döbber,Frank Kirschhöfer,Matthias Franzreb,Martina Pohl

doi:10.3389/fbioe.2018.00211

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.

Highlights

For the development of “green processes” on an industrial scale, there is a growing trend for continuous processes rather than batch reactions
The reaction kinetics depending on the actual substrate concentration is of interest, because the actual substrate concentration in the hydrogel immobilizates is assumed to be lower compared to the substrate solution due to mass transfer limitations
Method for the immobilization of enzymes in hydrogel lattices under mild conditions based on 3Dprinting

Summary

Introduction

For the development of “green processes” on an industrial scale, there is a growing trend for continuous processes rather than batch reactions. Afterwards, the hydrogel lattices were transferred into small batch reaction vials filled with the respective substrate solutions and the amount of the product formation over time was determined (see Figure 4). This hypothesis is tested, which shows the effectiveness factors of the enzymes entrapped in hydrogel plotted vs the theoretical apparent reaction rate constants.

Results

Conclusion