Molecular dynamics simulation guided distal mutation of Thermotoga naphthophila β-glucosidase for significantly enhanced synthesis of galactooligosaccharides and expanded product scope.

For efficient enzymatic production of health-beneficial galactooligosaccharides (GOSs), a glycone (-1)/aglycone (+2) subsite mutation strategy to engineer a thermophilic GH1 β-glucosidase (Tn0602) from Thermotoga naphthophila RKU-10 was introduced. Six single mutation variants (F226G, N246G, N246E, N222F, N222Y, G224T) and two double mutants (F226GF414S, F226GF414Y) were designed. The +2-subsite variant F226G produced 136 mM galactooligosaccharide 1.2-fold more than the wild type (115 mM). More significantly, a superimposed mutation of the -1/+2 subsites F226G/F414S gave a total GOS production of 314 mM (82.16% lactose conversion), 2.7-fold higher than the total GOS production of the wild type. Furthermore, the variant F226GF414S was profiled 241 mM of trisaccharide (galβ (1 → 3)/(1 → 4) lactose) and 73 mM tetrasaccharide (galβ (1 → 3)/(1 → 4) galβ (1 → 3)/(1 → 4) lactose). According to a 300-ns molecular dynamic simulation, the superimposed mutation increased GOS productivity and expanded the scope of products by changing the structural flexibility and reducing the steric hindrance of the substrate tunnel. Overall, our study successfully demonstrated that a - 1/+2 subsite mutagenesis method could be used in β-glucosidases Tn0602 to improve enzyme productivity and expand product scope, which could be a potential route to evolve retaining glycosidases towards the desired direction.