Engineering of ω-transaminase at binding pocket and access tunnel for preparation of (R)-1-phenoxypropan-2-amine.

Transaminase-catalyzed amination of prochiral ketones is regarded as a promising route for optically active amines production. These chiral amines serve as key building blocks in the synthesis of agrochemicals and pharmaceuticals. However, the limited substrate scope and substrate/(co)product inhibition have significantly hindered the industrial-scale implementation of transaminase-catalyzed processes. Engineering of TAs at binding sites is an efficient strategy to expand the capacity of binding pocket, thereby improving the catalytic efficiency. Furthermore, modification of access tunnel is crucial for adjusting the transfer efficiency of ligand. In this endeavor, we engineered an ω-TA from Nocardioides sp. CER19(NsTA) at binding sites and access tunnel, and applied the mutants for enantioselective synthesis of (R)-1-phenoxypropan-2-amine. Mutation at residues H62 and Y122 increased catalytic activity of the TA towards 1-phenoxyacetone, the optimal mutants NsTAH62A and NsTAY122A with improved enzyme activity was obtained, which was 2.0 and 1.5-fold higher than that of the wild-type NsTA respectively. This allowed completely conversion of 5 mM 1-phenoxyacetone to (R)-1-phenoxypropan-2-amine at 4 h and > 99 % ee (in comparison to 66 % convesion with the wild-type NsTA). Molecular dynamic simulation suggested improved flexibility of NsTAH62A, which may result in eliminating of substrate inhibition. While mutant NsTAD7 (with deletion of 7 amino acids at N-terminal) exhibited about 1.6-fold increase of conversion. Tunnel analysis suggested that reshaping of the tunnel entrance may alter the shape and size of the tunnel, which might promote the release of the products. These results of our study lay the foundation for enantioselective synthesis of (R)-1-phenoxypropan-2-amine and provide a promising engineering strategy to enhance the activity of TAs and overcome substrate/(co)product inhibition of ω-TA towards bulky substrates.