Improving enzymatic properties of BlTDH from Bacillus licheniformis through site-directed mutagenesis

L-threonine dehydrogenase (TDH) is a rate-limiting enzyme in the biosynthesis pathway of 2,5-dimethylpyrazine and 2,3,5-trimethylpyrazine, which are widely used food additives. However, natural TDH enzymes suffer from low catalytic activity and poor environmental adaptability, limiting their industrial applications. This study hypothesized that strategic site-directed mutagenesis of conserved amino acid residues within the substrate-binding pocket and catalytic domain could affect both enzymatic activity and environmental stability of TDH. To test this hypothesis, BlTDH from Bacillus licheniformis was selected as the target enzyme, and structure-oriented alanine substitution mutagenesis was systematically applied to five conserved residues (T94, H95, N157, T293, and G294). The results showed that among the five mutants (T94A, H95A, N157A, T293A and G294A), N157A mutant had a specific enzyme activity of 120.47 ± 1.88 mU/mg, which was 2.1 times higher than that of the wild-type. In addition, the N157A mutant showed better temperature stability and pH adaptability. Structural analysis revealed that the side chain volume of N157A mutant decreased, thereby expanding substrate binding space and reducing steric hindrance, which was conducive to the catalytic reaction. These findings validated the original hypothesis, demonstrating that rational amino acid substitutions can significantly affect the catalytic performance of TDH. The superior N157A mutant presented immediate commercial viability for industrial-scale production of food-grade pyrazine additives. Meanwhile, the established structure-activity relationship provides an engineering framework for optimizing the application of relevant NAD⁺-dependent dehydrogenases in biotechnology.