Endo- and exo-levanases from Bacillus subtilis HM7: Catalytic components, synergistic cooperation, and application in fructooligosaccharide synthesis.

Levan-type fructooligosaccharides (LFOS) exhibit significant biological activities and selectively promote the growth of certain beneficial bacteria. Levanase is an important enzyme for LFOS production. In this study, two isoforms of levanases, exo- and endo-type depolymerizing enzymes, from Bacillus subtilis HM7 isolated from Dynastes hercules larvae excrement were cloned, expressed, and characterized. The synergistic effect on the levan hydrolysis and kinetic properties of both isoforms were evaluated, indicating their cooperation in levan metabolism, where the endo-levanase catalyzes a rate-limiting step. In addition, homology models and molecular dynamics simulations revealed the key amino residues of the enzymes for levan binding and catalysis. It was found that both isoforms possessed distinct binding residues in the active sites, suggesting the importance of the specificity of the enzymes. Finally, we demonstrated the potential of endo-type levanase in LFOS synthesis using a one-pot reaction with levansucrase. Overall, this study fills the knowledge gap in understanding levanase's mechanism, making an important contribution to the fields of food science and biotechnology.