Immobilized Wild-Type and Mutant L‑Alanine Dehydrogenase from Thermus thermophilus Coupled with Formate Dehydrogenase for Asymmetric Synthesis of Noncanonical Amino Acids.

The asymmetric synthesis of noncanonical amino acids (ncAAs) is of increasing interest due to their importance as precursors for pharmaceuticals, enzyme inhibitors, and functional peptide building blocks. In this study, wild-type and Tyr92Ser mutant l-alanine dehydrogenases from Thermus thermophilus (TtAlaDH) (WT and Mut, respectively) were immobilized using three different strategies: covalent attachment on Eupergit C 250 L (EC250L), cross-linked enzyme aggregates (CLEAs), and entrapment in poly-(vinyl alcohol) (PVA) gel. Following a comparison of immobilization yield and recovered activity, the resulting preparations were characterized based on their optimum pH/temperature, thermal stability, and kinetic parameters. While covalent immobilization on EC250L and CLEA formation achieved high yields (>90%), they suffered from low recovered activity (≈5-12%). In contrast, PVA entrapment provided full immobilization yield while preserving near-native activity (95-100%). The WT and Mut preparations were individually coimmobilized with Chaetomium thermophilum formate dehydrogenase (CtFDH) in PVA gel to establish an integrated NADH regeneration system. The coimmobilized WT and Mut preparations enabled one-pot reductive amination of α-keto acids into enantiopure L-amino acids with excellent stereoselectivity (99% ee). Under optimized enzyme ratios and reaction conditions, l-alanine was obtained in at least 83% yield. Notably, the Mut preparations displayed enhanced selectivity toward bulkier substrates compared to the WT preparation, producing noncanonical l-norvaline and L-norleucine amino acids with yields of up to 47.5% and 74.8%, respectively. The coimmobilized PVA-Mut/CtFDH preparation retained high activity after multiple reuse cycles. This study provides a promising and scalable biocatalytic platform for the sustainable synthesis of l-alanine-derived noncanonical amino acids.