Improving the kinetic stability of a hyperthermostable β-mannanase by a rationally combined strategy

Feasible and easily accessible methods for the rational design of enzyme engineering strategies remain to be established. Thus, a new rationally combined strategy based on disulfide bond engineering and HotSpot Wizard 3.0 was proposed and experimentally demonstrated to be effective using a hyperthermostable β-mannanase. Ten of 42 mutants showed prominent enhancement of kinetic stability with 26.4%-39.9% increases in t_1/2 (75 °C) compared with the parent enzyme ManAKH. The best mutant, D273-V308, showed apparent increases in both optimal temperature (5 °C) and T₅₀ (6.8 °C), as well as advanced catalytic efficiency. The low rate of inactive mutants and the high rate of positive mutants indicated that newly introduced screening factors (distance from catalytic residues, Gibbs free energy term, molecular simulation, and visual inspections) greatly enhance the design of thermostable β-mannanase. Moreover, these findings further advance the industrial application of β-mannanase (ManAK) in food and food-related applications.