Rational Design of Flexible Loops Adjacent to the Active Site Enhances the Catalytic Efficiency of Bacillus β-Mannanase.

Mannan oligosaccharides (MOS) are valuable prebiotics, and enzymatic hydrolysis by β-mannanase is the preferred production route. Bacillus-derived GH26 β-mannanase (gmuG) holds industrial promise but suffers from low catalytic efficiency and weak pH stability. Here, we rationally engineered flexible loops near the active site via molecular dynamics, NMsim (normal mode-based geometric simulation), sequence conservation analysis, and MAESTRO prediction, and obtained the optimal double mutant M1 (E301K-T271V). Relative to the wild type, M1 showed 1.54-fold higher specific activity and nearly 2-fold improved pH stability at pH 8, with a nearly doubled kcat while retaining substrate affinity. Mechanistically, M1 enlarged the active site, shortened the attack distance of the catalytic residue, increased α-helix content, and rigidified neighboring loops. Fermentation optimization in Escherichia coli gave a maximum yield of 924.34 U/mL. This work offers an effective strategy for β-mannanase engineering and a high-performance biocatalyst for industrial MOS production.