Engineering an acid–base dyad into the peroxygenase-enabled mutant CYP199A4T252E yielded four in silico-designed double mutants, of which CYP199A4F182R/T252E showed the best dyad-like geometry and was characterized further. It delivered 10-fold higher initial H2O2-driven O-demethylation activity than wild type and CYP199A4T252E, alongside reduced catalase activity and improved peroxide utilization. However, it was more prone to H2O2-induced heme bleaching and rapid inactivation under standard dosing; slow, controlled H2O2 feeding sustained catalysis for hours. Overall, adding a second basic residue boosts per-oxy-gen-ase-like activity but reduces oxidative robustness, underscoring the trade-off between efficiency and peroxide tolerance and guiding future engineering of robust P450 peroxygenases.

Transesterification reactions are fundamental transformations in organic chemistry, yet performing them in aqueous media is challenging because of the competing hydrolysis reaction. In this study, we describe a mutant of alcohol oxidase from Phanerochaete chrysosporium (PcAOx-VPN) that also exhibits transesterification activity. Moreover, PcAOx-VPN displays no detectable hydrolytic activity, owing to its hydrophobic active site, which effectively excludes water. These characteristics make PcAOx-VPN a promising catalyst for transesterification reactions in aqueous media, a context that is typically compromised by competing hydrolysis.

Biophotoelectrocatalysis provides chemo-, regio- and stereoselective routes to chemicals by coupling redox biocatalysis with photoelectrocatalysis. This biomimetic strategy, however, is limited by unwanted photoelectrocatalysis side reactions and the high cost of redox mediators. Here we report a sustainable biosolar platform that uses bicarbonate (HCO3−), a hydrated form of CO2, as a redox mediator to drive oxyfunctionalization of inert C–H bonds. Using molybdenum-doped bismuth vanadate as a model photoelectrode, we accelerate two-electron H2O oxidation for in situ H2O2 production and mitigate enzyme-damaging OH· generation via HCO3− photoredox chemistry. Photoelectrochemical and spectroscopic analyses revealed that HCO3− directs the H2O oxidation pathway towards H2O2 through the formation of a peroxycarbonate intermediate at the photoanode surface. The integration of HCO3− mediation with H2O2-dependent unspecific peroxygenase achieves an exceptional turnover of various enantioselective C–H oxyfunctionalization reactions under ambient conditions. The HCO3−-mediated H2O2 photosynthetic system opens up opportunities for sustainable oxygenative biosynthesis. (Figure presented.)

The monoterpene citronellol often represents the substrate for the synthesis of other natural products and fragrances bearing α-branched tetrahydropyran moieties. In this contribution, we developed a process that combines in one-pot condition photocatalytic Schenck-ene reaction and biocatalytic halocyclization to synthesize enantiopure α-branched tetrahydropyrans starting from natural monoterpene citronellol. The reaction pathway of the enzymatic haloetherification, studied by combining experimental and theoretical studies, showed for the first time the key role played by the hydroperoxide functional group in the control of the regioselectivity of the cyclization step. Overall, a novel and sustainable synthetic procedure is reported as a new approach for α-branched tetrahydropyrans.

Peroxide-dependent enzymes often suffer from irreversible oxidative deactivation by the peroxide co-substrate. Transition metal mediated in situ generation of H2O2 offers continuous peroxide feeding in low concentration. However, free metal complexes often interact non-selectively with proteins, leading to mutual deactivation of metal catalysts and enzymes. Here, we report a spatial isolation strategy using zirconium-based metal-organic frameworks (UiO-67) to immobilize the transition metal catalytic unit [Cp*Rh(bpy)Cl]⁺. The porous MOF structure acts as a molecular sieve, excluding enzymes from the Rh sites on the framework, thus protecting both catalysts from mutual deactivation. The Rh modified UiO-67 (Rh@UiO-67) catalyzes the flavin-mediated electron transfer from formate to oxygen, generating H2O2 in a formate oxidase mimicking fashion. Its protein compatibility allows Rh@UiO-67 to fuel peroxyzymes for stable oxyfunctionalization. Compared to natural formate oxidase, this system also shows high stability to various pH and temperatures, enabling its application in versatile conditions.

Lignocellulosic wastes are naturally abundant carbon resources but have been underutilized due to their complex structure and recalcitrant nature. They require energy- and water-intensive processes, such as thermal, chemical, and/or mechanical pretreatments, for their valorization. Here, we report a new function of raw tree waste for driving the solar-powered oxygen reduction reaction (ORR) and biocatalytic oxyfunctionalization of hydrocarbons. We reveal that various lignocellulosic wastes, such as fallen leaves, waste wood, and wastepaper, can produce hydrogen peroxide (H2O2) using only O2, water, and light without any pretreatment. In particular, fallen leaves from Platanus trees exhibit high rates of ORR, which is ascribed to their superior photophysical properties, such as higher light extinction, longer charge relaxation lifetime, and lower electron transfer resistance. We treated the fallen leaves of Platanus with H2O2-dependent unspecific peroxygenase to produce optically pure alcohols and epoxides through the stereoselective hydroxylation and epoxidation of hydrocarbons. The waste-enzyme hybrid catalyst achieved record-high turnover frequency and total turnover number. This study establishes raw biomass wastes as green photocatalysts for sustainable photobiosynthesis, presenting a successful example of waste-to-wealth conversion.

Peroxygenases are promising biocatalysts for selective oxyfunctionalization reactions including hydroxylation, epoxidation, and sulfoxidation. In this study, we explore the activity of two recently reported peroxygenases from Collariella virescens (CviUPO) and Daldinia caldariorum (DcaUPO) in a range of synthetically relevant transformations. Both enzymes were heterologously expressed in Escherichia coli and tested for various oxidative reactions. DcaUPO generally demonstrated higher activity compared to CviUPO across several substrates, showing significant conversions in al-cohol and arene oxidations as well as enantioselective epoxidations of styrene derivatives. Notably, the enzymes exhibited complementary selectivities in several reactions including allylic hydroxylation and benzylic oxidation. These results broaden the substrate scope of CviUPO and DcaUPO and highlight their potential for industrial applications. However, challenges with enzyme expression in E. coli remain, necessitating future work on alternative expression systems such as Pichia pastoris to improve yields.

Hyaluronic acid (HA), a linear polysaccharide composed of alternating β-1,3-glucuronic acid (GlcA) and β-1,4-N-acetylglucosamine (GlcNAc) disaccharide units, is widely utilized in food, pharmaceutical, and cosmetic industries. Conventional in vitro HA biosynthesis is hindered by the reliance on costly nucleotide sugar precursors (UDP-GlcA and UDP-GlcNAc) and inefficient multienzyme coordination. To address these challenges, this study established a cell-free enzymatic cascade system integrating HA de novo synthesis with nucleotide recycling through eight pathway enzymes. By leveraging nucleotide sugar salvage pathways, UDP-GlcA and UDP-GlcNAc were efficiently synthesized from inexpensive monosaccharides, thereby bypassing energy-intensive de novo routes. Soluble expression of Pasteurella multocida hyaluronan synthase (PmHAS) was achieved by truncating its membrane-associated domains to enable sequential glycosyl transferase activity in aqueous systems. A dual ATP/UTP regeneration strategy was further implemented to sustain nucleotide supply, eliminating costly downstream purification. Under optimized conditions, the system produced 1.28 g/L HA within 24 h, with a molecular weight range of 1.28 × 10⁴to 1.02 × 10⁶Da and a substrate conversion yield of 65.9%. This work not only provides an economical platform for scalable HA synthesis but also establishes a modular enzymatic blueprint for engineering complex biopolymers, demonstrating broad applicability in synthetic glycobiology.

Fatty acids derived from renewable resources, such as vegetable oils, serve as essential feedstocks in various industries, including surfactants, cosmetics, lubricants, and polymers. Although current industrial applications of fatty acids are mainly centered around carboxylate group transformations, recent advancements in biocatalysis have opened new possibilities for converting fatty acids into valuable chemical building blocks. This contribution critically assesses novel biocatalytic transformations of fatty acids, including hydroxylation of the alkyl chain, epoxidation of C═C double bonds, reduction of the carboxylate group, and decarboxylation. These reactions hold great potential for producing important intermediates for chemical synthesis.

Practical Application: The enzymatic valorization of fatty acids offers transformative potential for sustainable oleochemical synthesis, presenting practical applications across various industries. Hydroxylated and epoxidized fatty acids are promising precursors for the production of polyesters, bio-based lubricants, and surfactants. Sophorolipids, derived from hydroxylated fatty acids, are gaining attraction as renewable biosurfactants with applications in detergents and cosmetics. Epoxidized fatty acids serve as intermediates for eco-friendly adhesives, coatings, and polymers. Furthermore, decarboxylation reactions yield alkanes, viable as biofuels, whereas reductions of carboxyl groups enable selective synthesis of aldehydes and alcohols for fragrances and pharmaceutical intermediates. The scalability of these biocatalytic transformations, combined with their mild operational conditions and high specificity, can substantially reduce environmental impact and production costs. These applications highlight biocatalysis as a pivotal technology for advancing the chemical industry toward sustainable practices.

Enzymatic processes for the remediation of wastewater containing organic pollutants are a promising alternative to advanced treatment processes that are often energy intensive and/or generate waste or by-products. For antibiotics, enzyme systems studied to date have been limited by substrate scope, pH tolerance, and stability. In this work, the remediation potential of two promiscuous H₂O₂-dependent enzymes is explored: the unspecific peroxygenase from Agrocybe aegerita (AaeUPO) and the chloroperoxidase from Curvularia inaequalis (CiVCPO), for the removal of four antibiotics commonly found in WWTP effluents and surface waters. While both enzymes showed a high removal potential for sulfamethoxazole (SMX) as a model antibiotic, CiVCPO was inactive in municipal wastewater, likely due to the presence of phosphate and nitrate. In contrast, AaeUPO remained active and stable within a suitable pH and temperature range. The transformation products showed decreased antibiotic activity against a susceptible strain of E. coli and decreased phytotoxicity, as indicated by the increased root length of Daucus carota. Peroxygenases are known to be sensitive to excess H₂O₂, and AaeUPO displays significant catalase activity at low substrate concentrations. To minimise H₂O₂-mediated inactivation, experiments were conducted at various H₂O₂ dosing rates in batch mode. Optimal conditions for the operation of a continuous enzymatic membrane reactor were then investigated, achieving over 95 % removal of SMX. This lays the groundwork for continuous operation and paves the way for efficient reactor design.

Composed of various biosourced metabolites, NaDES offer significant economic, health, and environmental benefits. Their remarkable ability to interact with target compounds through non-covalent bonds enhances their versatility. As solvents, excipients, cofactors, catalysts, solubilisation promoters, stabilisers, and absorption agents, NaDES provide distinct advantages over conventional substances and can even act as active compounds themselves. Furthermore, their role in advancing innovative synthesis and formulation strategies, particularly in nanotechnology and biotechnology, is driving research in these areas. This review is the first to explore all the potential applications of NaDES in the pharmaceutical industry, while taking a comprehensive look at the theory behind them. It gives a precise definition of NaDES and describes their composition, characteristics, molecular interactions, preparation, stability and recovery. It presents detailed applications in pharmaceutical synthesis, extraction and formulation, and discusses roles as active compounds or tools for innovation. Using green metrics, the efficiency of routes including NaDES is compared to that of conventional processes. Lastly, this review addresses often overlooked points such as toxicity and process limitations.

A photoelectrochemical (PEC) device induces electrochemical reactions on the surfaces of light-absorbing semiconductors to harness sunlight for producing valuable chemicals. The most critical issue in PEC devices is the poor stability of semiconductors in electrochemical environments. The stability can be enhanced by applying a transparent and conductive protection layer, which is usually prepared by an oxide thin film with tens of nanometers, on the semiconductor. Nevertheless, ensuring complete impermeability to an electrolyte remains a significant challenge because even a single pinhole in the thin film can lead to the dissolution of the entire underlying semiconductor layer. In this study, we present a facile and reliable protection method applicable to various semiconductors using a thick (200–500 μm) single crystal of TiO₂. The impermeability is ensured by the exceptionally high thickness, without compromising the device performance. We applied the protection layer on a halide perovskite semiconductor well-known for moisture instability, and inductively coupled plasma mass spectrometry rigorously confirmed that there was no dissolution of elements from the halide perovskite film. The robust protection layer also enabled the safe integration of the halide perovskite PEC device and a biocatalyst without concerns about Pb or halide toxicity. An old yellow enzyme from Thermus scotoductus (TsOYE) coupled with the PEC device enabled the trans-hydrogenation of the C 00000000 00000000 00000000 00000000 11111111 00000000 11111111 00000000 00000000 00000000 C bonds, demonstrating the expanded applicability and economic potential of PEC systems for producing fine chemicals and pharmaceutical intermediates.

Heme-containing unspecific peroxygenases (UPOs) have attracted significant attention as biocatalysts for oxidation reactions due to their ability to function without expensive nicotinamide cofactors. In the recent study, the UPO from aspergillus brasiliensis (AbrUPO) is found to catalyze the aromatic hydroxylation of substituted benzenes, a feature that distinguishes AbrUPO from other reported wild-type UPOs. To elucidate the underlying factors in the active site and substrate access channel of AbrUPO—which contains fewer phenylalanine residues compared to other UPOs that primarily catalyze benzylic hydroxylation—twenty two AbrUPO variants with single, double, triple, or quadruple amino acid substitutions were constructed to mimic the active sites or substrate access channels of other UPOs. A number of mutated variants exhibited altered activity and selectivity, and several positions were identified that influence enzyme chemoselectivity. Among them, substitution of alanine at position 186 with bulkier residues such as phenylalanine or leucine lead to a shift in chemoselectivity toward alkyl chain hydroxylation of substituted benzenes. Molecular docking studies indicated that the A186F mutation restricts the flexibility and reorientation of ethylbenzene in the active site of AbrUPO, thereby preventing oxidation at the aromatic ring while promoting benzylic hydroxylation.

In this study, we present a significant advancement in the field of enzymatic asymmetric reductive amination (ARA) of ketones, a pivotal reaction for chiral amine synthesis. Through a combination of semirational enzyme design and bioprocess development, we achieve the dual activation and stabilization of amine dehydrogenase (AmDH) to meet industrial demands. The engineered AmDH exhibits remarkable catalytic efficiency (turnover number, TON >1,000,000) and exceptional stability (half-life >7 days at 50 °C), with a broadened substrate scope including various aryl alkyl ketones and fatty ketones. Leveraging biobased oleic acid as an activator and stabilizer, we achieved kilogram-scale synthesis of chiral amines. Furthermore, the integration of AmDH with chemical catalysts in chemoenzymatic cascades has enabled the synthesis of a wide array of pharmaceutically relevant amines from diverse substrates, demonstrating the enzyme’s versatility and potential to transform synthetic chemistry.

Peroxygenases represent a class of versatile heme-thiolate enzymes capable of catalysing highly selective oxyfunctionalisation reactions, particularly the hydroxylation of non-activated C-H bonds. This transformation, which poses substantial challenges in conventional organic synthesis, underscores the potential of peroxygenases in green chemistry applications. While cytochrome P450 monooxygenases have long been the primary focus for such biocatalytic transformations, their industrial adoption has been limited due to complex electron transfer chains and cofactor requirements. In contrast, peroxygenases bypass these limitations by directly utilising hydrogen peroxide (H2O2) to activate the catalytic heme site, thereby circumventing the oxygen dilemma typically encountered in P450 catalysis. Key milestones in peroxygenase research include the identification of chloroperoxidase from Caldariomyces fumago and the subsequent discovery of unspecific peroxygenases, such as those from Agrocybe aegerita, which exhibit broad substrate specificity and high catalytic efficiency. Here, we explore the mechanistic pathway of peroxygenase-catalysed reactions, emphasising the formation and decay of Compound I and the catalytic cycle’s various functional outcomes. Critical aspects such as in situ H2O2 generation to mitigate enzyme inactivation, substrate loading strategies for practical applications, and the role of enzyme and reaction engineering in enhancing regio- and stereoselectivity are examined. Additionally, we address challenges in reaction scalability and operational stability for preparative-scale applications, offering insights into innovative protocols involving immobilised enzymes and non-aqueous reaction media. This review highlights recent advancements in the peroxygenase field and underscores the enzyme’s promising role in sustainable oxyfunctionalisation reactions.

Unspecific peroxygenases (UPOs) are highly versatile biocatalysts capable of removing various persistent environmental contaminants and performing sustainable chemical transformations. These oxidoreductases contain heme b as their prosthetic group. As all classical peroxidases, they are activated by the molecules of hydrogen peroxide to incorporate the oxygen atom into numerous organic molecules. Alternatively, they can use ascorbate as a cosubstrate. In sequence databases an ever-increasing number of their DNA and protein sequences occurs. Reconstructed molecular phylogeny of the corresponding peroxidase-peroxygenase superfamily reveals a high diversity of gene distribution for UPOs in the whole kingdom of fungi. A majority of identified UPO sequences stems from numerous species of Dikarya. Although members of this superfamily were recently detected also in early diverging fungal lineages, UPOs from the phyla of Mucoromycota, Glomeromycota and Chytridiomycota remain not sufficiently investigated. Moreover, newly discovered genes coding for UPOs were recently identified also among early diverging eukaryotic lineages of amoebas and green algae in various biotops. With a large palette of potential substrates these oxidoreductases serve as a versatile tool in enzyme catalysed synthetic reactions, but their real physiological substrates need to be recognized in the future. Most important among self-sufficient UPO-catalysed reactions are oxyfunctionalizations of various aliphatic and aromatic molecules. In this critical review an outlook is given for investigation and engineering of novel UPO variants including products of directed evolution. Future research on UPOs shall be mainly focused on basal fungal and emerging non-fungal sources for their promising applications in environmentally friendly technologies.

Plasma-generated H2O2 can be used to fuel biocatalytic reactions that require H2O2 as a cosubstrate, such as the conversion of ethylbenzene to (R)-1-phenylethanol ((R)-1-PhOl) catalyzed by unspecific peroxygenase from Agrocybe aegerita (rAaeUPO). Immobilization is recently shown to protect biocatalysts from inactivation by highly reactive plasma-produced species; however, H2O2 supply by the employed plasma sources (μAPPJ and DBD) is limiting for rAaeUPO performance. This study evaluates a recently introduced capillary plasma jet for suitability to supply H2O2 in situ. H2O2 production is modulated by varying the water concentration in the feed gas, providing a greater operating window for applications in plasma-driven biocatalysis. In a static system after 80 min of biocatalysis, a turnover number of 44,199 mol(R)-1-PhOl mol−1rAaeUPO is achieved without significant enzyme inactivation. By exchanging the reaction solution every 5 min, a total product yield of 122 μmol (R)-1-PhOl is achieved in 700 min run time, resulting in a total turnover number of 174,209 mol(R)-1-PhOl mol−1rAaeUPO. This study concludes that the capillary plasma jet, due to its flexibility regarding feed gas, admixtures, and power input, is well suited for in situ H2O2 generation for plasma-driven biocatalysis tailoring to enzymes with high H2O2 turnover.

The downstream product transformation of lignin depolymerization is of great interest in the production of high-value aromatic chemicals. However, this transformation is often impeded by chemical oxidation under harsh reaction conditions. In this study, we demonstrate that hypohalites generated in situ by the vanadium-containing chloroperoxidase from Curvularia inaequalis (CiVCPO) can halogenate various electron-rich and electron-poor phenol and phenolic acid substrates. Specifically, CiVCPO enabled decarboxylative halogenation, deformylative halogenation, halogenation, and direct oxidation reactions. The versatile transformation routes for the valorization of phenolic compounds showed up to 99% conversion and 99% selectivity, with a turnover number of 60,700 and a turnover frequency of 60 s^-1 for CiVCPO. This study potentially expands the biocatalytic toolbox for lignin valorization.

Engineering nonribosomal peptide synthetases (NRPSs) has been a “holy grail” in synthetic biology due to their modular nature and limited understanding of catalytic mechanisms. Here, we reported a computational redesign of the “gate-keeper” adenylation domain of the model NRPS-like enzyme carboxylic acid reductases (CARs) by using approximate mechanism-based geometric criteria and the Rosetta energy score. Notably, MabCAR3 mutants ACA-1 and ACA-4 displayed a remarkable improvement in catalytic efficiency (kcat/KM) for 6-aminocaproic acid, up to 101-fold. Furthermore, G418K exhibited an 86-fold enhancement in substrate specificity for adipic acid compared to 6-aminocaproic acid. Our work provides not only promising biocatalysts for nylon monomer biosynthesis but also a strategy for efficient NRPSs engineering.

This review analyzes a development in biochemistry, enzymology and biotechnology that originally came as a surprise. Following the establishment of directed evolution of stereoselective enzymes in organic chemistry, the concept of partial or complete deconvolution of selective multi-mutational variants was introduced. Early deconvolution experiments of stereoselective variants led to the finding that mutations can interact cooperatively or antagonistically with one another, not just additively. During the past decade, this phenomenon was shown to be general. In some studies, molecular dynamics (MD) and quantum mechanics/molecular mechanics (QM/MM) computations were performed in order to shed light on the origin of non-additivity at all stages of an evolutionary upward climb. Data of complete deconvolution can be used to construct unique multi-dimensional rugged fitness pathway landscapes, which provide mechanistic insights different from traditional fitness landscapes. Along a related line, biochemists have long tested the result of introducing two point mutations in an enzyme for mechanistic reasons, followed by a comparison of the respective double mutant in so-called double mutant cycles, which originally showed only additive effects, but more recently also uncovered cooperative and antagonistic non-additive effects. We conclude with suggestions for future work, and call for a unified overall picture of non-additivity and epistasis.