Characterization of thermostable and chimeric enzymes via isopeptide bond-mediated molecular cyclization

Mannooligosaccharides are released by mannan-degrading endo-β-1,4-mannanase and are known as functional additives in human and animal diets. To satisfy demands for biocatalysis and bioprocessing in crowed environments, in this study, we employed a recently developed enzyme-engineering system, isopeptide bond-mediated molecular cyclization, to modify a mesophilic mannanase from Bacillus subtilis. The results revealed that the cyclized enzymes showed enhanced thermostability and ion stability, resilience to aggregation and freeze-thaw treatment by maintaining their conformational structures. Additionally, by using the SpyTag/SpyCatcher system, we generated a mannanase-xylanase bifunctional enzyme that exhibited a synergistic activity in substrate deconstruction without compromising substrate affinity. Interestingly, the dual-enzyme ring conformation was observed to be more robust than the linear enzyme but inferior to the single-enzyme ring conformation. Taken together, these findings provided new insights ...