A Biotin-Man-manno-cyclophellitol activity-based probe (ABP) was used to pull-down putative mannanases from the secretome of the parental strain of A. niger MA234.1 grown for at least 72h after transfer on konjac flour (kF). AnManA was successfully enriched in the secretome. Total protein identification of the same secretome did not reveal the presence of another well characterized mannanase form A. niger: AnMan26A Both recombinant AnManA and AnMan26A were expressed in A. niger and purified. The recombinant proteins were labelled with either a Man-manno-cyclophellitol or a Man-man-manno-cyclophellitol probe. The m/z of the complex between the probe and the tryptic peptide containing the active-site nucleophile of the two mannanases was successfully identified. This way the mechanism-based activity of the developed mannanase ABPs was confirmed.