Site-directed mutagenesis to enhance thermostability of Caulobacter sp. D5 ω-transaminase for efficient bioamination of biobased aldehydes

To enhance the application potential of ω-transaminase ATA1012 in the efficient bioamination of biobased aldehydes, this study performed site-directed mutagenesis at key sites in the active center and flexible loop region, resulting in the mutant Q25F with significantly improved thermostability. The half-life of Q25F increased from 4.2 h to 25.1 h at 37 °C and from 0.6 h to 3.3 h at 50 °C. Benefiting from its enhanced stability, Q25F efficiently converted lignin-derived vanillin (260 mM) to vanillylamine within 12 h, achieving a yield of 92.6% and a selectivity >99%. Kinetic analysis revealed a Km of 0.2 mM for vanillin, a kcat of 8.9 s −1 , and a catalytic efficiency (kcat/Km) of 33.1 M −1 s −1 . Furthermore, by optimizing the mutagenesis strategy, engineered strains capable of efficiently catalyzing the transamination of biomass-derived furfural (FAL) and 5-hydroxymethylfurfural (HMF) into biobased amines were constructed. This study establishes a site-directed mutagenesis approach for enhancing the thermostability of ω-transaminase, providing an effective route for the high-value bioconversion of carbohydrates and lignin in lignocellulosic biomass.