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.

Structural and functional catalytic characteristics of cross-linked enzyme aggregates (CLEA) are reviewed. Firstly, advantages of enzyme immobilization and existing types of immobilization are described. Then, a wide description of the factors that modify CLEA activity, selectivity and stability is presented. Nowadays CLEA offers an economic, simple and easy tool to reuse biocatalysts, improving their catalytic properties and stability. This immobilization methodology has been widely and satisfactorily tested with a great variety of enzymes and has demonstrated its potential as a future tool to optimize biocatalytic processes.

Suicide inactivation of peroxidases by hydrogen peroxide is the major deterrent to using such biocatalysts in oxidative processes. This has been successfully addressed by the in situ generation of H 2 O 2 . In this study, we have developed a novel multi-enzyme biocatalyst that has been immobilized on agarose-type carriers to oxidize phenols using oxygen and formic acid as indirect oxidants. This original system couples the in situ production of H 2 O 2 to the phenol oxidation via an enzymatic cascade that involved three different enzymes (formate dehydrogenase, NADH-oxidase and peroxidase) and two different redox cofactors: nicotinamide and flavin derivatives. The cascade reaction only works when enzymes are immobilized on the solid support since soluble enzymes are dramatically inactivated under the reaction conditions. The highest oxidation efficiency was achieved by combining two different solid biocatalysts:(1) formate dehydrogenase and NADH-oxidase co-immobilized onto agarose beads activated with glyoxyl groups and (2) peroxidase immobilized onto agarose beads as well but activated with boronate groups. Unlike conventional peroxidase-mediated oxidations with exogenous H 2 O 2 , this novel system enables the quantitative oxidation of phenol without the addition of H 2 O 2 . Furthermore, this novel system can use a broad range of redox cofactors with similar oxidative effectiveness. Therefore, this novel immobilized trienzyme system removes important pollutants such as hydroxylated aromatic derivatives ( phenol, 4-aminophenol, 2,4-dichloro-phenol or α-naphthol) using formic acid and molecular oxygen as substrates. In addition, this system generates CO 2 as waste beyond the oxidized phenols that can be easily separated from the aqueous solution. The sustainability of this system is supported by an E-factor of 1.3 and an atom economy of 43%. † Electronic supplementary information (ESI) available. See