Engineering of a novel ω‑transaminase for efficient whole‑cell catalytic synthesis of vanillylamine

ω-Transaminases (ω-TAs) have been widely applied in synthesis of chiral amines. However, they are often constrained by low activity and poor stability in organic media during the conversion of non-natural substrates such as vanillin. In this study, a novel ω-transaminase (XPTA) with high activity was identified to enable efficient bioamination of vanillin to vanillylamine, a pivotal precursor for synthesis of various high-value compounds. Subsequent semi-rational design enhanced the catalytic efficiency ( k cat /K m ) of mutant M1 6.93-fold compared to the wild-type XPTA, along with significantly improved thermal stability and substrate specificity towards vanillin. These enhanced catalytic performances were confirmed from the optimized substrate-binding conformation and strengthened hydrogen-bond networks. Furthermore, a petroleum ether–water (1:9, vol/vol) biphasic system was developed to facilitate whole-cell catalytic synthesis of vanillylamine. Using (S)-(-)-α-methylbenzylamine (S-MBA) as the amine donor at an equimolar ratio of vanillin, vanillylamine yield achieved 98.7%. An efficient and green biocatalytic pathway for vanillylamine synthesis is thus established, integrating enzyme mining, semi‑rational engineering, biphasic reaction, and equimolar chiral amine donation as a system‑level strategy for bio‑based aromatic chemical production.