Recombinant l‐Amino Acid Oxidase with Broad Substrate Spectrum for Co‐substrate Recycling in (S)‐Selective Transaminase‐Catalyzed Kinetic Resolutions

Chiral and enantiopure amines can be produced by enantioselective transaminases via kinetic resolution of amine racemates. This transamination reaction requires stoichiometric amounts of co‐substrate. A dual‐enzyme recycling system overcomes this limitation: l‐amino acid oxidases (LAAO) recycle the accumulating co‐product of (S)‐selective transaminases in the kinetic resolution of racemic amines to produce pure (R)‐amines. However, availability of suitable LAAOs is limited. Here we use the heterologously produced, highly active fungal hcLAAO4 with broad substrate spectrum. H2O2 as byproduct of hcLAAO4 is detoxified by a catalase. The final system allows using sub‐stoichiometric amounts of 1 mol% of the transaminase co‐substrate as well as the initial application of l‐amino acids instead of α‐keto acids. With an optimized protocol, the synthetic potential of this kinetic resolution cascade was proven at the preparative scale (>90 mg) by the synthesis of highly enantiomerically pure (R)‐methylbenzylamine (>99 %ee) at complete conversion (50 %). An efficient co‐substrate recycling system consisting of a heterologous expressed and highly active l‐amino acid oxidase combined with a catalase was applied for kinetic resolutions to produce enantiopure (R)‐amines in ω‐(S)‐transaminase catalyzed reactions. Consequently, catalytic instead of stoichiometric amounts of co‐substrate can be applied in preparative scale reactions.