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.

合理设计酶工程策略的可行且容易获得的方法仍有待建立。因此，提出了一种新的基于二硫键工程和HotSpot Wizard 3.0的合理组合策略，并实验证明了使用超热β-甘露聚糖酶是有效的。与亲本酶ManAKH相比，在42个突变体中有10个显示出显着的动力学稳定性增强，t _1/2（75°C）时提高了26.4％–39.9％。最佳突变体D273–V308显示最佳温度（5°C）和T ₅₀均明显增加（6.8°C），以及先进的催化效率。无活性突变体的低发生率和阳性突变体的高发生率表明，新引入的筛选因子（与催化残基的距离，吉布斯自由能项，分子模拟和肉眼检查）大大增强了热稳定β-甘露聚糖酶的设计。而且，这些发现进一步促进了β-甘露聚糖酶（ManAK）在食品和食品相关应用中的工业应用。