Engineering CBM of β-Mannanase from Paenibacillus to Alter Product Chain Length for Preparing Mannan Oligosaccharide Prebiotics.

Biomass polysaccharides are explored as inexpensive raw materials for preparing value-added products, such as mannan oligosaccharides (MOS). However, the varied composition of MOS and the underlying mechanism have not been studied thoroughly yet. Here a β-mannanase from Paenibacillus sp. was engineered for hydrolyzing biomass polysaccharides and producing prebiotic MOS. Structural analysis and alanine scanning revealed two hot spots, Q66 and T88, in the carbohydrate binding module (CBM). The mutant T88Y demonstrated a 65% activity improvement and a 5-fold thermal stability enhancement at 70 °C, and Q66F produced 2.2 g/L of mannobiose and mannotriose from locust bean gum, with a 46% activity increase. Moreover, Q66F generated a 2.0 molar ratio of mannobiose/mannotriose, and a reverse ratio of 0.4 was found in Q66R. The application of Q66R was further validated in the fermentation of citrus peel waste. The results provided efficient biocatalysts for producing prebiotic MOS from biomass polysaccharides and deeper comprehension of altering product chain length of β-mannanases from CBM.