biostudies-literatureUnknown11Liu XL-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 <i>Bacillus licheniformis</i> 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 resulFood chemistry. Molecular sciences100272https://www.ebi.ac.uk/biostudies/studies/S-EPMC12276384biostudies-literatureImproving enzymatic properties of BlTDH from &lt;i&gt;Bacillus licheniformis&lt;/i&gt; through site-directed mutagenesis.PMC12276384Liu XLi HTang ZGu HChen SQuan WfalseImproving enzymatic properties of BlTDH from &lt;i&gt;Bacillus licheniformis&lt;/i&gt; 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 <i>Bacillus licheniformis</i> 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 resul2025-01-01T00:00:00Z2025 Dec2026-03-27T16:18:25.67Z2025-08-30T03:05:59.65ZS-EPMC122763844068737910.1016/j.fochms.2025.100272