Mutational Analysis and Application of a Multidomain GH157 Family Endo-β-1,3-glucanase from Bacteroides sp. M27

Endo-β-1,3-glucanases play a crucial role in food processing and biological control. This study characterized the function and structure of the glycoside hydrolase (GH) 157 family β-1,3-glucanase (BsGlc157A) from Bacteroides. Structural analysis of BsGlc157A revealed that it is a multidomain enzyme, containing a GH157 family catalytic domain at the N-terminus and two potential carbohydrate-binding modules (CBMs) at the C-terminus. Substrate affinity experiments had shown that these two C-terminus domains can specifically bind insoluble β-1,3-glucan (Curdlan). Additionally, homology analysis indicated that these two CBMs were associated with the CBM81 and CBM43 families, respectively. The interactions between the GH157 family catalytic domain and substrates were analyzed and confirmed through site-directed mutagenesis. Furthermore, the calculated mutation energy was used to generate two mutants, G60A and N180A, which showed a 1.2- and 1.6-fold increase in specific activity, respectively. BsGlc157A-N180A was shown to effectively hydrolyze Curdlan, yielding β-glucooligosaccharides (degree of polymerization 2-6) with a soluble oligosaccharide production of 68.8% through synergistic hydrolysis. This study not only provides insight into the catalytic mechanism of endo-β-1,3-glucanases in the GH157 family but also contributes to the molecular modification and potential applications of β-1,3-glucanases.