Analysis of the galactomannan binding ability of β-mannosidases, BtMan2A and CmMan5A, regarding their activity and synergism with a β-mannanase

[Display omitted] •BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2.•BtMan2A displayed stronger irreversible binding to galactomannan than CmMan5A.•BtMan2A binding to galactomannan did not affect its activity, while CmMan5A lost activity.•BtMan2A binding was pH-dependent, with increased binding ability at lower pH.•CmMan5A synergised with CcManA, while BtMan2A did not – even though the enzyme was active.•High loadings of BtMan2A abolished CcManA activity; at protein ratios ≥ 5:1. Both β-mannanases and β-mannosidases are required for mannan-backbone degradation into mannose. In this study, two β-mannosidases of glycoside hydrolase (GH) families 2 (BtMan2A) and 5 (CmMan5A) were evaluated for their substrate specificities and galactomannan binding ability. BtMan2A preferred short manno-oligomers, while CmMan5A preferred longer ones; DP >2, and galactomannans. BtMan2A displayed irreversible galactomannan binding, which was pH-dependent, with higher binding observed at low pH, while CmMan5A had limited binding. Docking and molecular dynamics (MD) simulations showed that BtMan2A galactomannan binding was stronger under acidic conditions (-8.4 kcal/mol) than in a neutral environment (-7.6 kcal/mol), and the galactomannan ligand was more unstable under neutral conditions than acidic conditions. Qualitative surface plasmon resonance (SPR) experimentally confirmed the reduced binding capacity of BtMan2A at pH 7. Finally, synergistic β-mannanase to β-mannosidase (BtMan2A or CmMan5A) ratios required for maximal galactomannan hydrolysis were determined. All CcManA to CmMan5A combinations were synergistic (≈1.2-fold), while combinations of CcManA with BtMan2A (≈1.0-fold) yielded no hydrolysis improvement. In conclusion, the low specific activity of BtMan2A towards long and galactose-containing oligomers and its non-catalytic galactomannan binding ability led to no synergy with the mannanase, making GH2 mannosidases ineffective for use in cocktails for mannan degradation.