Molecular modification and preliminary application of a novel mannanase from Alkalihalobacillus hemicellulosilyticus

Mannooligosaccharides (MOS) are high-quality prebiotic components, and enzymatic production of MOS is the most efficient, simple, and environmentally friendly method. Mannanase is the key enzyme involved in the decomposition of mannan for MOS production. However, there are currently challenges in obtaining thermally stable Mannanase. In this study, a novel thermo-alkaline mannanase, ManB085, was discovered from Alkalihalobacillus hemicellulosilyticus and heterologously expressed. The optimal temperature and pH of this enzyme are 75 °C and 10, respectively, and it remains stable at pH 7-12 and temperatures below 50 °C. Through rational design, a mutant ManB085M, with the non-catalytic domain truncated, was obtained, showing significantly improved thermal stability. The half-life of the mutant at 75 °C is 12.3 times that of the wild type, and the T₅₀ increased by 23 °C. Notably, the enzyme can also produce MOS by hydrolyzing hemicellulose in coffee grounds. To enhance the MOS production capability, a Carbohydrate-Binding Module (CBM) was added to the C-terminus of ManB085M, significantly improving its ability to extract MOS from coffee grounds. This study proposes a novel mannanase with significant potential in MOS production.