LAUSR

Mannan is an important renewable resource whose backbone can be hydrolyzed by β-mannanases to generate manno-oligosaccharides of various sizes. Only a few glycoside hydrolase (GH) 113 family β-mannanases have been functionally and structurally characterize. Here, we report the function and structure of a novel GH113 β-mannanase from Bacillus sp. N16-5 (BaMan113A). BaMan113A exhibits a substrate preference toward manno-oligosaccharides and releases mannose and mannobiose as main hydrolytic products. The crystal structure of BaMan113A suggest that the enzyme shows a semi-enclosed substrate-binding cleft and the amino acids surrounding the +2 subsite form a steric barrier to terminate the substrate-binding tunnel. Based on these structural features, we conducted mutagenesis to engineer BaMan113A to remove the steric hindrance of the substrate-binding tunnel. We found that F101E and N236Y variants exhibit increased specific activity toward mannans comparing to the wild-type enzyme. Meanwhile, the product profiles of these two variants toward polysaccharides changed from mannose to a series of manno-oligosaccharides. The crystal structure of variant N236Y was also determined to illustrate the molecular basis underlying the mutation. In conclusion, we report the functional and structural features of a novel GH113 β-mannanase, and successfully improved its endo-acting activity by using structure-based engineering.