Aromatic dioxygenases (ADOs) catalyse the oxidative cleavage of CC bonds in aromatic olefins, producing valuable aldehydes or ketones. Due to their coenzyme independence, ADOs are attractive catalysts. However, the limited catalytic performance of natural ADOs restricts their broader practical application. Here, we determined the 2.2 Å crystal structure of an ADO from Coniochaeta pulveracea (CpuADO). Structure-guided mutagenesis targeting residues near the active site identified a mutant F349W, which exhibits enhanced catalytic efficiency (k_cat/K_m) toward sinapyl alcohol by about 1.41-fold compared to the wild-type enzyme. In parallel, AI-based computational screening identified a mutant W338D, which shows improved catalytic efficiency for several aromatic olefins, including 1.3-fold for coniferyl alcohol, 1.7-fold for 4-vinylguaiacol, and approximately 12-fold for isoeugenol. Molecular dynamics (MD) simulations revealed stabilised Fe²⁺–CC distances (approximately 5.5 Å in F349W and 4.7–6.2 Å in W338D) and reduced structural fluctuations, indicating improved substrate positioning. These findings provide a structure-based strategy for engineering ADOs with enhanced catalytic performance toward lignin-related aromatic olefins, allowing for more efficient lignin valorisation.

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.

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.

Alcohol oxidases (AOxs) catalyze the aerobic oxidation of alcohols to the corresponding carbonyl products (aldehydes or ketones), producing only H₂O₂ as the byproduct. The majority of known AOxs, however, have a strong preference for small, primary alcohols, limiting their broad applicability, e.g., in the food industry. To broaden the product scope of AOxs, we performed structure-guided enzyme engineering of a methanol oxidase from Phanerochaete chrysosporium (PcAOx). The substrate preference was extended from methanol to a broad range of benzylic alcohols by modifying the substrate binding pocket. A mutant (PcAOx-EFMH) with four substitutions exhibited improved catalytic activity toward benzyl alcohols with increased conversion and k_cat toward the benzyl alcohol from 11.3 to 88.9% and from 0.5 to 2.6 s^-1, respectively. The molecular basis for the change of substrate selectivity was analyzed by molecular simulation.

The realm of photobiocatalytic alkane biofuel synthesis has burgeoned recently; however, the current dearth of well-established and scalable production methodologies in this domain remains conspicuous. In this investigation, we engineered a modified form of membrane-associated fatty acid photodecarboxylase sourced from Micractinium conductrix (McFAP). This endeavour resulted in creating an innovative assembled photoenzyme-membrane (protein load 5 mg cm⁻²), subsequently integrated into an illuminated flow apparatus to achieve uninterrupted generation of alkane biofuels. Through batch experiments, the photoenzyme-membrane exhibited its prowess in converting fatty acids spanning varying chain lengths (C6–C18). Following this, the membrane-flow mesoscale reactor attained a maximum space-time yield of 1.2 mmol L⁻¹ h⁻¹ (C8) and demonstrated commendable catalytic proficiency across eight consecutive cycles, culminating in a cumulative runtime of eight hours. These findings collectively underscored the photoenzyme-membrane's capability to facilitate the biotransformation of diverse fatty acids, furnishing valuable benchmarks for the conversion of biomass via photobiocatalysis.

In the present work we propose a bienzymatic cascade for the oxyfunctionalisation of cycloalkanes to cyclic alcohols/cyclic ketones. By combining a H₂O₂-dependent peroxygenase with a O₂-consuming and H₂O₂-producing alcohol oxidase an overall aerobic oxidation system was established. A convincing proof-of-concept is presented and some current limitations are outlined.

Aromatic aldehydes are important aromatic compounds for the flavour and fragrance industry. In this study, a parallel cascade combining aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) and unspecific peroxygenase from the basidiomycete Agrocybe aegerita (AaeUPO) to convert aromatic primary alcohols into high-value aromatic aldehydes is proposed. Key influencing factors in the process of enzyme cascade catalysis, such as enzyme dosage, pH and temperature, were investigated. The universality of PeAAOx coupled with AaeUPO cascade catalysis for the synthesis of aromatic aldehyde flavour compounds from aromatic primary alcohols was evaluated. In a partially optimised system (comprising 30 μM PeAAOx, 2 μM AaeUPO at pH 7 and 40 °C) up to 84% conversion of 50 mM veratryl alcohol into veratryl aldehyde was achieved in a self-sufficient aerobic reaction. Promising turnover numbers of 2800 and 21,000 for PeAAOx and AaeUPO, respectively, point towards practical applicability.

Unspecific peroxygenases (UPOs) represent an emerging class of catalysts for the selective oxyfunctionalisation of C–H- and C = C groups. Until now, only a few UPOs have been characterised. In this study, we report a new peroxygenase identified from the Unspecific Peroxygenase Database. The UPO from Agaricus bisporus var. bisporus (AbvbUPO) has been heterologously expressed in Aspergillus niger and initially characterised with respect to its basic biochemical features. Furthermore, its catalytic properties were evaluated with enzymatic cascade reactions of choline oxidase (AnChOx) and AbvbUPO, which the AnChOx provided H₂O₂ necessary via reductive activation of oxygen in situ. Three types of oxyfunctionalizations, such as hydroxylation of ethylbenzene, epoxidations of styrene and cyclohexene, sulfoxidations of methyl phenyl sulfide and phenyl vinyl sulfide, were successfully achieved. We also investigated the activity of AbvbUPO on fatty acids in some more detail. The experimental results show that Under the above conditions, AbvbUPO had the higher activity for cyclohexene epoxidation and sulfonation of sulfide substrates. The concentration of epoxy cyclohexane was 2.91 mM, and the concentration of methyl phenyl sulfoxide was 3.69 mM. The regioselectivity of AbvbUPO was ω-1 bonds position of linear saturated fatty acid. All in all, AbvbUPO exhibits some interesting differences which may put the basis for further understanding of the factors determining peroxygenase selectivity.

Hydrocarbons are essential base chemicals as energy carriers and starting materials for chemical manufacture. So-called fatty acid photodecarboxylases (FAPs) represent interesting catalysts for the conversion of natural fatty acids into hydrocarbons thereby giving access to alkanes from renewable feedstock. Today, however, only few FAPs are known. In the current study we report a new FAP from the marine organism Micractinium conductrix (McFAP). In contrast to currently known FAPs McFAP exhibits high catalytic activity towards short and medium fatty acids. Recombinant expression and basic biochemical characterisation of this new member of the FAP family is reported.

Carvol and carvone are oxidation products from the natural product limonene. They are important raw materials for the flavours and fragrances industry and also act as pharmaceutical active ingredients. Orange waste peels possibly represent an attractive source for limonene, but studies on valorizing orange peel wastes are rare. In this study, we report a new enzymatic cascade system for the in-situ conversion of limonene from orange peel into valued-added carvol and carvone. The use of deep eutectic solvents (DES) allows for efficient in-situ extraction of limonene from waste orange peels. We propose a dual function use of DES as solvent for the extraction and the biocatalytic oxidation of limonene as well as cosubstrate to promote the oxidation reaction. Using ChCl-Pro-H₂O DES for the extraction of limonene from waste orange peels, approximately 17 milligrams of limonene per gram of orange peel was achieved at 40°C for 24 h. Then, with ChCl-Pro-H₂O DES as the extractant and reaction medium, a cascade reaction system of choline oxidase (ChOx) and unspecific peroxygenase (UPO) was established to catalyse the conversion of limonene into carvol and carvone. The concentration of the final products was up to about 1.6 mmol L⁻¹. This study showed a biocatalytic transformation pathway and provides technical support for the high-value utilization of waste in orange peel.

Hydrocarbon synthesis from (waste)oils enabled by a cascade of lipase-catalysed hydrolysis and decarboxylase-catalysed decarboxylation has become an active area of research en route to alternative, biobased fuels. However, Poor substrate transport efficiency is a major issue causing low reaction rates. This study focused on a protein self-assembly strategy based on SpyTag/SpyCatcher to overcome diffusion limitations. For this, two fusion proteins, TLL-Linker-SpyCatcher based on the lipase from Thermomyces lanuginosus and CvFAP-Linker-SpyTag based on the fatty acid photodecarboxylase from Chlorella variabilis were designed. A covalent multi-enzyme complex (TLL-CvFAP) was formed spontaneously by self-assembly of each enzyme. The effects of temperature, pH and molar ratio of self-assembled components on assembly efficiency were investigated. The results showed that the multi-enzyme complex TLL-CvFAP reached about 60% after 12 h of assembly, and the enzyme activity of the multienzyme complex was increased by about 50% compared to that of the corresponding non-assembled enzymes. Under optimized conditions 10 mM soybean oil were converted into 25 mM of the corresponding hydrocarbons, suggesting a good potential of biofuel synthesis.

Phthalic acid esters (PAEs) are widely used as plastic additives to increase the flexibility and durability of plastics. Constantly leaching out from plastics, PAEs are ubiquitously found in the environment. As PAEs exhibit biological activities such as being endocrine disruptive, the quest for efficient degradation strategies continues. Here, we report a bienzymatic degradation system for PAEs to phthalic acid (PA) using a cascade comprising two hydrolases, EstJ6 and P8219. The reaction conditions were optimized with respect to concentrations of both enzymes, temperature, and initial pH. Finally, the substrate scope of the new cascade was investigated, revealing that particularly PAEs with relatively small alcohols were degraded to more than 90 %. This present study provides a potential doable biocatalytic strategy for the complete hydrolysis of PAEs.

Unspecific peroxygenases (UPOs, EC 1.11.2.1) are promising oxyfunctionalization catalysts because of their unique stereoselectivity. However, so far only a few UPOs have been reported. In this study, gene mining was used to identify a gene from Galerina marginata that coded for a novel UPO (GmaUPO). GmaUPO was expressed in Pichia pastoris X-33 by scale-up fermentation (the UPO activity of the culture supernatant was 118 U/L). GmaUPO exhibited a molecular weight of 40 kDa and exhibited highest activity at 35°C and pH 9, respectively. Furthermore, GmaUPO was demonstrated to catalyze the epoxidation, sulfoxidation, and hydroxylation of common substrates, particularly fatty acids such as tridecanoic acid. The molecular basis for GmaUPO regioselectivity for fatty acid hydroxylation was explored by molecular modelling. The regioselectivity was mostly governed by the architecture of the enzyme's active site.

A bienzymatic cascade for selective sulfoxidation is presented. The evolved recombinant peroxygenase from Agrocybe aegeritra catalyses the enantioselective sulfoxidation of thioanisole whereas the choline oxidase from Arthrobacter nicotianae provides the H2 O2 necessary via reductive activation of ambient oxygen. The reactions are performed in choline chloride-based deep eutectic solvents serving as co-solvent and stoichiometric reductant at the same time. Very promising product concentrations (up to 15 mM enantiopure sulfoxide) and catalyst performances (turnover numbers of 150,000 and 2100 for the peroxygenase and oxidase, respectively) have been achieved.

A photobiocatalytic cascade transforming natural triglycerides into alkanes/alkenes is proposed. Starting from natural triglycerides, free fatty acids have been obtained using lipases. The free fatty acids were then, in a photoenzymatic step, decarboxylated into the C1-shortened alkanes using a recently described photodecarboxylase from Chlorella variabilis NC64A. This cascade produced alkanes from various natural (waste) oils in significant amounts (up to 24 g L⁻¹) and may provide a basis for valorisation of waste oils into a next generation of biodiesel.

Poor H ₂ O ₂ -resistance by enzymes is a key bottleneck in the epoxidation process of oil by enzymatic methods. In this study, the stability of three lipases, from Aspergillus oryzae lipase (AOL), Aspergillus fumigatus lipase B (AflB), and marine Janibacter (MAJ1), in the presence of H ₂ O ₂ was evaluated in different types of natural deep eutectic solvents (NADES). This stability was strengthened significantly in the NADES compared to the buffer. Specifically, AOL retained 84.7% of its initial activity in the presence of choline chloride/sorbitol (1:1 M ratio) and 3 mol L ⁻¹ H ₂ O ₂ after 24 h incubation at 40°C. In addition, the two-phase epoxidation process was optimized with AOL in ChCl/sorbitol to reach up to 96.8% conversion under the optimized conditions (molar ratio of octanoic acid/H ₂ O ₂ /C=C-bonds = 0.3:1.5:1, enzyme loading of 15 U g ⁻¹ of soybean oil, ChCl/sorbitol content of 70.0% of the weight of hydrophilic phase, and reaction temperature of 50°C). Moreover, the lipase dispersed in NADES retained approximately 66% of its initial activity after being used for seven batch cycles. Overall, NADES-based enzymatic epoxidation is a feasible and promising strategy for the synthesis of epoxidized oils.

The use of natural deep eutectic solvents (NADES) as multifunctional solvents for limonene bioprocessing was reported. NADES were used for the extraction of limonene from orange peel wastes, as solvent for the chemoenzymatic epoxidation of limonene, and as sacrificial electron donor for the in situ generation of H ₂ O ₂ to promote the epoxidation reaction. The proof-of-concept for this multifunctional use was provided, and the scope and current limitations of the concept were outlined.

Natural deep eutectic solvents (NADES) are proposed as alternative solvents for peroxygenase-catalysed oxyfunctionalization reactions. Choline chloride-based NADES are of particular interest as they can serve as solvent, enzyme-stabiliser and sacrificial electron donor for the in situ H₂O₂ generation. This report provides the first proof-of-concept and basic characterisation of this new reaction system. Highly promising turnover numbers for the biocatalysts of up to 200,000 have been achieved.