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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 and 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 into the mechanisms of molecular cyclization on stability improvement and will be useful in the production of new functional oligosaccharides and feed additives.

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