Reshaping the binding channel of a novel GH113 family β-mannanase from Paenibacillus cineris (PcMan113) for enhanced activity

Abstract Endo-β-mannanases are important enzymes for degrading lignocellulosic biomass to generate mannan, which has significant health effects as a prebiotic that promotes the development of gut microbiota. Here, a novel endo-β-mannanase belonging to glycoside hydrolase (GH) family 113 from Paenibacillus cineris (PcMan113) was cloned, expressed and characterized, as one of only a few reported GH113 family β-mannanases. Compared to other functionally and structurally characterized GH113 mannanases, recombinant PcMan113 showed a broader substrate spectrum and a better performance. Based on a structural homology model, the highly active mutant PcMT3 (F110E/N246Y) was obtained, with 4.60- and 5.53-fold increases of enzyme activity (towards KG) and catalytic efficiency (k cat/K m, against M5) compared with the WT enzyme, respectively. Furthermore, molecular dynamics (MD) simulations were conducted to precisely explore the differences of catalytic activity between WT and PcMT3, which revealed that PcMT3 has a less flexible conformation, as well as an enlarged substrate-binding channel with decreased steric hindrance and increased binding energy in substrate recognition. In conclusion, we obtained a highly active variant of PcMan113 with potential for commercial application in the manufacture of manno-oligosaccharides. Graphical Abstract