Abstract: Baeyer-Villiger monooxygenase from Pseudomonas aeruginosa (PaBVMO) selectively inserts an oxygen atom into the unconventional side of the carbonyl group of 10-carbonyl stearic acid to produce octyl sebacate (which can be used to synthesize material monomers sebacic acid and 8-hydroxyoctanoic acid). In order to break through the dual bottleneck of low catalytic efficiency and significant substrate inhibition of PaBVMO at high substrate concentrations in sebacic acid biosynthesis, this study proposed a semi-rational remodeling strategy for the enzyme's substrate channel. Through the analysis of enzyme-substrate binding free energy and substrate force, the key gating loop structure of the substrate channel was resolved. Based on this strategy, the optimal mutant PaBVMO_G398A obtained in this study gave a catalytic turnover number (TON) of 3.45×10⁴, which is 6.3 times higher than that of the parent, and 1.7 times higher than that of the parental enzyme. Meanwhile, the inactivation half-life is prolonged from 1.87 h to 6.14 h, and the stability is significantly enhanced. Gaussian-accelerated molecular dynamics (GaMD) simulations were used to elucidate the synergistic mechanism of G398A mutation inducing the rearrangement of the active pocket of PaBVMO_G398A, which in turn induced the decrease in the substrate inhibition of PaBVMO_G398A by 10-carbonyl stearic acid, the increase in affinity for FAD and the increase in stability.