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.

Biocatalysis is a key technology enabling plastic recycling. However, despite advances done in the development of plastic-degrading enzymes, the molecular mechanisms that govern their catalytic performance are poorly understood, hampering the engineering of more efficient enzyme-based technologies. In this work, we study the hydrolysis of PET-derived diesters and PET trimers catalyzed by the highly promiscuous lipase B from Candida antarctica (CALB) through QM/MM molecular dynamics simulations supported by experimental Michaelis–Menten kinetics. The computational studies reveal the role of the pH on the CALB regioselectivity toward the hydrolysis of bis-(hydroxyethyl) terephthalate (BHET). We exploit this insight to perform a pH-controlled biotransformation that selectively hydrolyzes BHET to either its corresponding diacid or monoesters using both soluble and immobilized CALB. The discoveries presented here can be exploited for the valorization of BHET resulting from the organocatalytic depolymerization of PET.

Multi-enzymatic cascades with enzymes arranged in close-proximity through a protein scaffold can trigger a substrate channeling effect, allowing for efficient cofactor reuse with industrial potential. However, precise nanometric organization of enzymes challenges the design of scaffolds. In this study, we create a nanometrically organized multi-enzymatic system exploiting engineered Tetrapeptide Repeat Affinity Proteins (TRAPs) as scaffolding for biocatalysis. We genetically fuse TRAP domains and program them to selectively and orthogonally recognize peptide-tags fused to enzymes, which upon binding form spatially organized metabolomes. In addition, the scaffold encodes binding sites to selectively and reversibly sequester reaction intermediates like cofactors via electrostatic interactions, increasing their local concentration and, consequently, the catalytic efficiency. This concept is demonstrated for the biosynthesis of amino acids and amines using up to three enzymes. Scaffolded multi-enzyme systems present up to 5-fold higher specific productivity than the non-scaffolded ones. In-depth analysis suggests that channeling of NADH cofactor between the assembled enzymes enhances the overall cascade throughput and the product yield. Moreover, we immobilize this biomolecular scaffold on solid supports, creating reusable heterogeneous multi-functional biocatalysts for consecutive operational batch cycles. Our results demonstrate the potential of TRAP-scaffolding systems as spatial-organizing tools to increase the efficiency of cell-free biosynthetic pathways.

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 H₂O₂. 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 H₂O₂ 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 H₂O₂, this novel system enables the quantitative oxidation of phenol without the addition of H₂O₂. Furthermore, this novel system can use a broad range of redox cofactors with similar oxidative effectiveness. Therefore, this novel immobilized tri-enzyme 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 CO₂ 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%.