Enzymolysis for effective grain processing: Computer-aided optimization of a 1,3-1,4-β-glucanase with improved...

β-Glucanases, widely applied in grain processing, are commonly restricted for efficient industrial application due to the limited thermostability. In this study, a 1,3-1,4-β-glucanase (BisGlu16B_ΔC) was optimized for thermostability through a computer-aided design of energy optimization. Three variants (T40K, Q53L, and S311Y) were selected and generated a combined mutant T40K/Q53L/S311Y (M3). Comparing with the WT, M3 exhibited better thermostability (with t_1/2 at 60 °C extend by 126 min), higher specific activity (1.24 folds; 69,700 vs. 56,200 U/mg), higher catalytic effciency (1.18 folds; 14,100 vs. 11,900 mL‧s^-1‧mg^-1), and improved protease resistance. For mechanism, more hydrogen bonds, salt bridges, and rigid secondary components in M3 led to an enhanced overall rigidity, boosting the thermostability. While enhanced long-range negative interactions affected some key residues in the catalytic channels, improving the catalytic efficiency. For application, M3 showed superiority with higher dry matter digestibility (1.49 folds; 80.3 % vs. 53.9 %) in simulated gastrointestinal system, together with more reduction of filtration time (1.55 folds; 22.2 % vs. 14.3 %) and viscosity (2.37 folds; 10.2 % vs. 4.3 %) during malting, comparing with the WT. Furthermore, the strongest synergistic effects were found between xylanase and M3, among all β-glucanases tested. All results verified M3 as an efficient β-glucanase for grain processing industry.