Overcoming Biochemical Limitations of Galactose Oxidase through the Design of a Solid-Supported Self-Sufficient Biocatalyst.

Galactose Oxidase (GalOx) has gained significant interest in biocatalysis due to its ability for selective oxidation beyond the natural oxidation of galactose, enabling the production of valuable derivatives. However, the practical application of GalOx has been hindered by the limited availability of active and stable biocatalysts, as well as the inherent biochemical limitations such as oxygen (O2 ) dependency and the need for activation. In this study, we addressed these challenges by immobilizing GalOx into agarose-based and Purolite supports to enhance its activity and stability. Additionally, we identified and quantified the oxygen supply limitation into solid catalysts by intraparticle oxygen sensing showing a trade-off between the amount of protein loaded onto the solid support and the catalytic effectiveness of the immobilized enzyme. Furthermore, we coimmobilized a heme-containing protein along with the enzyme to function as an activator. To evaluate the practical application of the immobilized GalOx, we conducted the oxidation of galactose in an instrumented aerated reactor. The results showcased the efficient performance of the immobilized enzyme in the 8 h reaction cycle. Notably, the GalOx immobilized into dextran sulfate-activated agarose exhibited improved stability, overcoming the need for a soluble activator supply, and demonstrated exceptional performance in galactose oxidation. These findings offer promising prospects for the utilization of GalOx in technical biocatalytic applications.

Biocatalysis in Continuous-Flow Microfluidic Reactors.

The implementation of continuous-flow transformations in biocatalysis has received remarkable attention in the last few years. Flow microfluidic reactors represent a crucial technological tool that has catalyzed this trend by promising tremendous improvement in biocatalytic processes across a host of different levels, including bioprocess development, intensification of reactions, implementation of new methods of reaction screening, and enhanced reaction scale-up. However, the full realization of this promise requires a synergy between these biocatalytic reaction features and the design and operation of microfluidic reactors. Here an overview on the different applications of flow biocatalysis is provided according to the format of the enzyme used: free vs immobilized form. Until now, flow biocatalysis has been implemented on a case-by-case approach but challenges and limitations are discussed in order to be overcome, and making continuous-flow microfluidic reactors as universal tool a reality.

Chemical Reaction Engineering to Understand Applied Kinetics in Free Enzyme Homogeneous Reactors.

Chemical reaction engineering is interested in elucidating the reaction kinetics through the determination of the fundamental influencing variables. The understanding of enzyme kinetics is needed to implement the potential of enzymes to satisfy determined production targets and for the design of the reactor. The quantification of the enzyme kinetics is implemented by the elucidation and building of the kinetic model (it includes one or more kinetic equations). In the context of process development, the kinetic model is not only useful to identify feasibility and for optimizing reaction conditions but also, at an early stage of development it is very useful to anticipate implementation bottlenecks, and so guide reactor setup. In this chapter we describe theoretical and practical considerations to illustrate the methodological framework of kinetic analysis. We take as study cases four archetypal kinetic cases by using as example the hydrolysis of cellobiose catalyzed by a beta-glucosidase. We show the different experimental data that can be obtained by the monitoring of enzymatic reactions in different configuration of free enzyme homogeneous ideal reactors; we show step-by-step the visualization, treatment, and analysis of data to elucidate kinetic models and the procedure for the quantification of kinetic constants. Finally, the performance of different reactors is compared in the interplay with the enzyme kinetics. This book chapter aims at being useful for a broad multidisciplinary audience and different levels of academic development.