Structure-guided engineering of L-aspartate-α-decarboxylase for enhanced substrate tolerance and β-alanine yield

β-Alanine is an important precursor for food additives, drugs, and nitrogen-containing compounds, and its biosynthesis is mainly catalyzed by L-aspartate-α-decarboxylase (PanD). However, the inherent limitations of wild-type PanD, including low catalytic efficiency and pronounced substrate inhibition at high concentrations, significantly constrain its industrial applications. This study systematically compared three PanD orthologs (EcPanD from Escherichia coli, CgPanD from Corynebacterium glutamicum, and BsPanD from Bacillus subtilis), revealing BsPanD's superior catalytic efficiency. BsPanD exhibited substrate inhibition at high L-aspartate concentrations, with conversion rates of 78 % and 52 % at 60 g/L and 100 g/L substrate, respectively. Through the construction of protein structures and reasonable structure-oriented design, as well as computational screening and experimental verification, we selected five mutants (BsPanD^T4W, BsPanD^I33A, BsPanD^P40N, BsPanD^I88W, BsPanD^N109E) with significantly improved tolerance from 40 candidate mutants. These mutants achieved 90.6–92.9 % conversion rates at 60 g/L substrate, yielding 36.4–37.4 g/L β-alanine. In addition, kinetic analyses demonstrated reduced K_m values (28–45 % decrease) in all mutants, indicating enhanced substrate affinity. Notably, BsPanD^I33A exhibited the highest catalytic efficiency (k_cat/K_m = 1.95 s⁻¹ mmol⁻¹ L, a 57.3 % increase over the wild-type), attributed to reduced steric hindrance from the Ile33Ala mutation. With its exceptional performance at high substrate concentration (92.9 % conversion at 60 g/L) and optimized catalytic efficiency, BsPanD^I33A emerges as the most promising biocatalyst for industrial β-alanine production. These results provide novel insights into PanD catalysis and establish a robust platform for industrial β-alanine biosynthesis.