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he hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. Here, we have engineered a highly regioselective fungal peroxygenase for the w-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward sub-terminal hydroxylation with a drop in the over-oxidation activity. While crystallographic soaking experiments and molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was produced by Pichia pastoris at 0.4 g/L in a fed-batch bioreactor and used in the preparative synthesis of 1.4 g of (w-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee through the (S)-enantiomer.

Chiral sulfoxides are valuable building blocks in asymmetric synthesis. However, the biocatalytic synthesis of chiral sulfoxides is still challenged by low product titres. Herein, we report the use of peroxygenase as a catalyst for asymmetric sulfoxidation under non-aqueous conditions. Upon covalent immobilisation, the peroxygenase showed stability and activity under neat reaction conditions. A large variety of sulfides was converted into chiral sulfoxides in very high product concentration with moderate to satisfactory optical purity (e. g. 626 mM of (R)-methyl phenyl sulfoxide in approx. 89 % ee in 48 h). Further polishing of the ee value via cascading methionine reductase A (MsrA) gave>99 % ee of the sulfoxide. The robustness of the enzymes and high product titer is superior to the state-of-the-art methodologies. Gram-scale synthesis has been demonstrated. Overall, we demonstrated a practical and facile catalytic method to synthesize chiral sulfoxides., BT/Biocatalysis

Non-thermal plasmas are used in various applications to inactivate biological agents or biomolecules. A complex cocktail of reactive species, (vacuum) UV radiation and in some cases exposure to an electric field together cause the detrimental effects. In contrast to this disruptive property of technical plasmas, we have shown previously that it is possible to use non-thermal plasma-generated species such as H2O2 as cosubstrates in biocatalytic reactions. One of the main limitations in plasma-driven biocatalysis is the relatively short enzyme lifetime under plasma-operating conditions. This challenge could be overcome by immobilizing the enzymes on inert carrier materials. Here, we tested whether immobilization is suited to protect proteins from inactivation by plasma. To this end, using a dielectric barrier discharge device (PlasmaDerm), plasma stability was tested for five enzymes immobilized on ten different carrier materials. A comparative analysis of the treatment times needed to reduce enzyme activity of immobilized and free enzyme by 30% showed a maximum increase by a factor of 44. Covalent immobilization on a partly hydrophobic carrier surface proved most effective. We conclude from the study, that immobilization universally protects enzymes under plasma-operating conditions, paving the way for new emerging applications., BT/Biocatalysis

Biocatalytic oxidation reactions of toluene derivates to the corresponding aldehydes are typically challenged by regio- and chemoselectivity issues. In this contribution we address both challenges by a combined reactant- and reaction engineering approach. We demonstrate that the peroxygenase-catalysed transformation of ring-substituted toluenes proceeds highly regioselectively in benzylic position. Furthermore, neat reaction conditions not only enable attractive product concentrations (up to 185 mM) but also result in highly chemoselective oxidations to the aldehyde level., BT/Biocatalysis

The hydroxylation of fatty acids is an appealing reaction in synthetic chemistry, although the lack of selective catalysts hampers its industrial implementation. In this study, we have engineered a highly regioselective fungal peroxygenase for the ¿-1 hydroxylation of fatty acids with quenched stepwise over-oxidation. One single mutation (A77L, Fett variant) found by structural studies and saturation mutagenesis near the Phe catalytic tripod narrowed the heme cavity, promoting a dramatic shift toward subterminal hydroxylation with a drop in the over-oxidation activity as determined by GC/MS. While molecular dynamic simulations shed light on this unique oxidation pattern, the selective biocatalyst was overexpressed in Pichia pastoris at 0.4 g/L-1 operating a fed-batch bioreactor and used for the preparative synthesis of 1.4 g of (¿-1)-hydroxytetradecanoic acid with 95% regioselectivity and 83% ee for the S-enantiomer.