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

ABSTRACT

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. H2 O2 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 %).