Ene-reductases from the old yellow enzyme (OYE) family have been traditionally employed in the reduction of conjugated C═C double bonds. This study explores the underutilized oxidative potential of OYEs, demonstrating their capability to catalyze the enantioselective desaturation of carbonyl compounds. Utilizing a deprotonated tyrosine residue as a catalytic base, we developed a method to enable OYE-catalyzed desaturation at ambient temperature and alkaline pH without the need for high-temperature conditions. Through screening of various OYE enzymes, we identified several candidates from different genera with enhanced desaturase activity across different substrates. This work broadens the scope of biocatalytic applications for OYEs, introducing a novel approach to the synthesis of chiral α,β-unsaturated carbonyl compounds.@en

Vicinal halohydrins are key building blocks to produce bioactive molecules and drugs, especially if they can be obtained in enantiomerically pure form. In this study, we present a bi-enzymatic sequence that allows to obtain vic-halohydrins through a photochemoenzymatic olefin hydroxy halogenation followed by a lipase catalysed kinetic resolution. The absolute configuration of the resulting products was determined using Mosher's method.@en

An enzymatic method for the selective hydroxylation of phenols using a peroxygenase from Aspergillus brasiliensis (AbrUPO) is reported. A broad range of phenolic starting materials can be selectively transformed into the corresponding hydroquinones. Semi-preparative syntheses of several hydroquinones were realised without further optimization pointing out the applicability of this enzyme as biocatalyst.

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Hydrogels that can disintegrate upon exposure to reactive oxygen species (ROS) have the potential for targeted drug delivery to tumor cells. In this study, we developed a diphenylalanine (FF) derivative with a thioether phenyl moiety attached to the N-terminus that can form supramolecular hydrogels at neutral and mildly acidic pH. The thioether can be oxidized by ROS to the corresponding sulfoxide, which makes the gelator hydrolytically labile. The resulting oxidation and hydrolysis products alter the polarity of the gelator, leading to disassembly of the gel fibers. To enhance ROS sensitivity, we incorporated peroxizymes in the gels, namely, chloroperoxidase CiVCPO and the unspecific peroxygenase rAaeUPO. Both enzymes accelerated the oxidation process, enabling the hydrogels to collapse with 10 times lower H2O2 concentrations than those required for enzyme-free hydrogel collapse. These ROS-responsive hydrogels could pave the way toward optimized platforms for targeted drug delivery in the tumor microenvironment.

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Utilisation of fatty acids generally relies on pre-existing functional groups such as the carboxylate group or C=C-double bonds. Addition of new functionalities into the hydrocarbon part opens up new possibilities for fatty acid valorisation. In this contribution we demonstrate the synthetic potential of a peroxygenase mutant AaeUPO−Fett for selective fatty acid oxyfunctionalisation. The ω-1 hydroxy fatty acid (esters) produced are further transformed into lactones, alcohols, esters and amines via multi-enzyme cascades thereby paving the way for new fatty acid valorisation pathways.

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Heme-dependent oxygenases (i.e. P450 monooxygenases and peroxygenases) are highly selective catalysts for the selective oxyfunctionalisation or organic compounds. Both enzyme classes exhibit mechanistic similarities (i.e. using so-called compound I (CpdI) as active oxidation species) and differences in how CpdI is formed. From the differences also practical differences arise which may influence the scalability, economic attractiveness and environmental impact of P450 monooxygenase- or peroxygenase-catalysed reactions. In this contribution we propose a range of performance indicators to compare the potential of both enzyme classes.

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Mol-scale oxyfunctionalization of cyclohexane to cyclohexanol/cyclohexanone (KA-oil) using an unspecific peroxygenase is reported. Using AaeUPO from Agrocybe aegerita and simple H2O2 as an oxidant, cyclohexanol concentrations of more than 300 mM (>60% yield) at attractive productivities (157 mM h-1, approx. 15 g L-1 h-1) were achieved. Current limitations of the proposed biooxidation system have been identified paving the way for future improvements and implementation.

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Unspecific peroxygenases (UPOs) are promising biocatalysts for oxyfunctionalisation reactions, owing to their simplicity of handling, stability and robustness. A limitation of using UPOs on a large scale is their deactivation in the presence of even rather modest concentrations of H₂O₂, requiring a constant and controlled supply of low amount of H₂O₂. Herein, we report an organometallic complex [Cp*Ir(pica)NO₃] {pica=picolinamidate=κ²-pyridine-2-carboxamide ion (−1)} 1 capable of efficiently regenerating FMNH₂ from FMN (TOF=350 h⁻¹, 298 K), driven by NaHCOO; FMNH₂, in turn, spontaneously reacts with O₂ leading to H₂O₂. After having studied the compatibility of 1 with the UPO from Agrocybe aegerita (rAaeUPO PaDa-I) and individuated the best experimental conditions, we applied such a hybrid catalytic tandem in some hydroxylation, epoxidation and sulfoxidation reactions. Best performances were obtained by using a 1/rAaeUPO molar ratio of 50. TONs for the biocatalyst of up to 18933 were obtained for the transformation of ethylbenzene derivatives into (R)-1-phenylethanols (ee>99 %). 1/rAaeUPO was found to oxidise also cis-methyl styrene (TON=13488), leading exclusively (1R,2S)-cis-methyl styrene oxide (ee>99 %), cyclohexane (TON=1634) and thioanisole (TON=1369).

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Heat is a fundamental feedstock, where more than 80% of global energy comes from fossil-based heating process. However, it is mostly wasted due to a lack of proper techniques of utilizing the low-quality waste heat (<100 °C). Here we report thermoelectrobiocatalytic chemical conversion systems for heat-fueled, enzyme-catalyzed oxyfunctionalization reactions. Thermoelectric bismuth telluride (Bi₂Te₃) directly converts low-temperature waste heat into chemical energy in the form of H₂O₂ near room temperature. The streamlined reaction scheme (e.g., water, heat, enzyme, and thermoelectric material) promotes enantio- and chemo-selective hydroxylation and epoxidation of representative substrates (e.g., ethylbenzene, propylbenzene, tetralin, cyclohexane, cis-β-methylstyrene), achieving a maximum total turnover number of rAaeUPO (TTN_rAaeUPO) over 32000. Direct conversion of vehicle exhaust heat into the enantiopure enzymatic product with a rate of 231.4 μM h⁻¹ during urban driving envisions the practical feasibility of thermoelectrobiocatalysis.

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A photochemoenzymatic halodecarboxylation of ferulic acid was achieved using vanadate-dependent chloroperoxidase as (bio)catalyst and oxygen and organic solvent as sole stoichiometric reagents in a biphasic system. Performance and selectivity were improved through a phase transfer catalyst, reaching a turnover number of 660.000 for the enzyme.

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The use of water-miscible organic co-solvents in biocatalysis is a simple procedure for obtaining higher enzymatic activities toward hydrophobic substrates. However, effects on activity and stability have to be carefully evaluated, also with regard to the type and concentration of the respective co-solvent. In this contribution, we investigated and evaluated the effect of some common water-miscible co-solvents on the biocatalytic performance of the recombinant unspecific peroxygenase rAaeUPO from Agrocybe aegerita. rAaeUPO showed promising activities in the presence of high concentrations of the best co-solvent acetonitrile, which enabled to use higher substrate concentrations (≥100 mM). Employing high acetonitrile concentrations for UPO-mediated oxidation of ethylbenzene to (R)-1-phenylethanol was demonstrated under preparative scale conditions and led to product accumulation rates of 31 mM h−1.@en

The pulp and paper manufacturers generate approximately 50 million metric tons of lignin per annum, most of which has been abandoned or incinerated because of lignin's recalcitrant nature. Here, we report bias-free photoelectrochemical (PEC) oxidation of lignin coupled with asymmetric hydrogenation of C=C bonds. The PEC platform consists of a hematite (α-Fe2O3) photoanode and a silicon photovoltaic-wired mesoporous indium tin oxide (Si/mesoITO) photocathode. We substantiate a new function of photoelectroactivated α-Fe2O3 to extract electrons from lignin. The extracted electrons are transferred to the Si/mesoITO photocathode for regenerating synthetic nicotinamide cofactor analogues (mNADHs). We demonstrate that the reduction kinetics of mNAD+s depend on their reduction peak potentials. The regenerated mNADHs activate ene-reductases from the old yellow enzyme (OYE) family, which catalyze enantioselective reduction of α,β-unsaturated hydrocarbons. This lignin-fueled biocatalytic PEC system exhibits an excellent OYE's turnover frequency and total turnover number for photobiocatalytic trans-hydrogenation through cofactor regeneration. This work presents the first example of PEC regeneration of mNADHs and opens up a sustainable route for bias-free chemical synthesis using renewable lignin waste as an electron feedstock.

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Non-enantioselective alcohol dehydrogenases (ADHs) are rarely found in the biocatalysis portfolio. Generally, highly enantioselective ADHs are sought for. Using such ADHs for the oxidation of racemic alcohols generally results in a kinetic resolution of the starting material, which is unfavourable if the ketone represents the product of interest. In the current contribution we report the ADH from Sphingobium yanoikuyae (SyADH) as non-enantioselective ADH for the complete oxidation or rac-heptan-2-ol (representing further 2-alkanols).@en

Cytochromes P450 catalyze oxidation of chemically diverse compounds and thus offer great potential for biocatalysis. Due to the complexity of these enzymes, their dependency of nicotinamide cofactors and redox partner proteins, recombinant microbial whole cells appear most appropriate for effective P450-mediated biocatalysis. However, some drawbacks exist that require individual solutions also when P450 whole-cell catalysts are used. Herein, we compared wet resting cells and lyophilized cells of recombinant E. coli regarding P450-catalyzed oxidation and found out that lyophilized cells are well-appropriate as P450-biocatalysts. E. coli harboring CYP105D from Streptomyces platensis DSM 40041 was used as model enzyme and testosterone as model substrate. Conversion was first enhanced by optimized handling of resting cells. Co-expression of the alcohol dehydrogenase from Rhodococcus erythropolis for cofactor regeneration did not affect P450 activity of wet resting cells (46% conversion) but was crucial to obtain sufficient P450 activity with lyophilized cells reaching a conversion of 72% under the same conditions. The use of recombinant lyophilized E. coli cells for P450 mediated oxidations is a promising starting point towards broader application of these enzymes.

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