Enzyme cofactors play a major role in biocatalysis, as many enzymes require them to catalyze highly valuable reactions in organic synthesis. However, the cofactor recycling is often a hurdle to implement enzymes at the industrial level. The fabrication of heterogeneous biocatalysts co‐immobilizing phosphorylated cofactors (PLP, FAD+, and NAD+) and enzymes onto the same solid material is reported to perform chemical reactions without exogeneous addition of cofactors in aqueous media. In these self‐sufficient heterogeneous biocatalysts, the immobilized enzymes are catalytically active and the immobilized cofactors catalytically available and retained into the solid phase for several reaction cycles. Finally, we have applied a NAD+‐dependent heterogeneous biocatalyst to continuous flow asymmetric reduction of prochiral ketones, thus demonstrating the robustness of this approach for large scale biotransformations.

We expanded the application of self-sufficient heterogeneous biocatalysts containing coimmobilized w-transaminases and pyridoxal 5´-phosphate (PLP) to efficiently operate packed-bed reactors in continuous flow. Using a w-transaminase from Halomonas elongata co-immobilized with PLP onto porous methacrylate-based carriers coated with polyethyleneimine, we operated a packed-bed reactor continuously for up to 50 column volumes at 1.45 mL x min -1 in the enantioselective deamination of model amines (α-methylbenzyl amine), yielding > 90% conversion in all cycles without exogenous addition of cofactor. In this work, we expanded the concept of self-sufficient heterogeneous biocatalysts to other w-transaminases such as the ones from Chromobacterium violaceum and Pseudomonas fluorescens. We found that enzymes with lower affinities towards PLP present lower operational stabilities in flow, even when coimmobilizing PLP. Furthermore, w-transaminases co-immobilized with PLP were successfully implemented for the continuous synthesis of amines and the sustainable metrics were assessed.These results give some clues to exploit PLP-dependent w-transaminases under industrially relevant continuous operations in a more cost-effective and environmentally friendly process.

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The integration of enzyme-catalyzed reactions in flow systems has been boosted during the last few years. Flow chemistry has been proposed in modern synthetic chemistry as a technology for process...

The development of cell-free and self-sufficient biocatalytic systems represents an emerging approach to address more complex synthetic schemes under nonphysiological conditions. Herein, we report the development of a self-sufficient heterogeneous biocatalyst for the synthesis of chiral alcohols without the need to add an exogenous cofactor. In this work, an NADPH-dependent ketoreductase was primarily stabilized and further co-immobilized with NADPH to catalyze asymmetric reductions without the addition of an exogenous cofactor. As a result, the immobilized cofactor is accessible, and thus, it is recycled inside the porous structure without diffusing out into the bulk, as demonstrated by single-particle in operando studies. This self-sufficient heterogeneous biocatalyst was used and recycled for the asymmetric reduction of eleven carbonyl compounds in a batch reactor without the addition of exogenous NADPH to achieve the corresponding alcohols in 100 % yield and >99 % ee; this high performance was maintained over five consecutive reaction cycles. Likewise, the self-sufficient heterogeneous biocatalyst was integrated into a plug flow reactor for the continuous synthesis of one model secondary alcohol, which gave rise to a space-time yield of 97-112 g L day ; additionally, the immobilized cofactor accumulated a total turnover number of 1076 for 120 h. This is one of the few examples of the successful implementation of continuous reactions in aqueous media catalyzed by cell-free and immobilized systems that integrate both enzymes and cofactors into the solid phase.