A protein engineering approach for expanding the substrate scope of the (S)‐selective Chromobacterium violaceum amine transaminase is presented. Amino acid residues in the small binding pocket of the active site were targeted in order to increase the pocket size for acceptance of primary amines bearing two bulky groups. A highly sensitive fluorescence assay was then used to evaluate the generated enzyme variants for their activity towards propyl‐ and benzyl‐substituted screening substrates. The best variant, L59A/F88A, was successfully applied in the kinetic resolution of 1,2‐diphenylethylamine using different conditions and substrate loadings. The variant L59A/F88A generated enantiomerically pure (R)‐1,2‐diphenylethylamine with ee>99% under all tested conditions. The variant also holds great promise for synthesis of hydrophobic compounds as it shows optimum activity when 20‐30% (v/v) DMSO is applied as cosolvent. The variant L59A/F88A provides a great addition to the available catalyst toolbox for synthesis of chiral amines, as it is the first published (S)‐selective amine transaminase showing activity towards benzyl‐substituted primary amines.

One of the main factors hampering the implementation in industry of transaminase-based processes for the synthesis of enantiopure amines is their often low storage and operational stability. Our still limited understanding of the inactivation processes undermining the stability of wild-type transaminases represents an obstacle to improving their stability through enzyme engineering. In this paper we present a model describing the inactivation process of the well-characterized (S)-selective amine transaminase from Chromobacterium violaceum. The cornerstone of the model, supported by structural, computational, mutagenesis and biophysical data, is the central role of the catalytic lysine as a conformational switch. Upon breakage of the lysine-PLP Schiff base, the strain associated with the catalytically active lysine conformation is dissipated in a slow relaxation process capable of triggering the known structural rearrangements occurring in the holo-to-apo transition and ultimately promoting dimer dissociation. Due to the occurrence in the literature of similar PLP-dependent inactivation models valid for other non-transaminase enzymes belonging to the same fold-class, the role of the catalytic lysine as conformational switch might extend beyond the transaminase enzyme group and offer new insight to drive future non-trivial engineering strategies.

Dynamic kinetic resolution (DKR) reactions in which a stereoselective enzyme and a racemization step are coupled in one pot would represent powerful tools for the production of enantiopure amines through enantioconvergence of racemates. The exploitation of DKR strategies is currently hampered by the lack of effective, enzyme‐compatible and scalable racemization strategies for amines. In the present work, the proof of concept of a fully biocatalytic method for amine racemization is presented. Both enantiomers of the model compound 1‐methyl‐3‐phenylpropylamine could be racemized in water and at room temperature using a couple of wild‐type, non‐proprietary, enantiocomplementary amine transaminases and a minimum amount of pyruvate/alanine as a co‐substrate couple. The biocatalytic simultaneous parallel cascade reaction presented here poses itself as a customizable amine racemization system with potential for the chemical industry in competition with traditional transition‐metal catalysis.