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

Both ß-mannanases and ß-mannosidases are required for mannan-backbone degradation into mannose. In this study, two ß-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 ß-mannanase to ß-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.

Time dependence of enzyme synergism during the degradation of model and natural lignocellulosic substrates.

Cellulosic ethanol production relies on the biochemical (enzymatic) conversion of lignocellulose to fermentable sugars and ultimately to bioethanol. However, the cost of lignocellulolytic enzymes is a limiting factor in the commercialisation of this technology. This therefore necessitates the optimisation of lignocellulolytic enzyme cocktails through the elucidation of synergistic interactions between enzymes so as to improve lignocellulose hydrolysis and also lower protein loadings in these reactions. However, many factors affect the synergism that occurs between these lignocellulolytic enzymes, such as enzyme ratios, substrate characteristics, substrate loadings, enzyme loadings and time. This review examines the effect of time on the synergistic dynamics between lignocellulolytic enzymes during the hydrolysis of both complex (true) lignocellulosic substrates and model substrates. The effect of sequential and simultaneous application of the lignocellulolytic enzymes on the synergistic dynamics during the hydrolysis of these substrates is also explored in this review. Finally, approaches are further proposed for efficient and synergistic hydrolysis of both complex lignocellulosic substrates and model substrates. With respect to the synergistic enzymatic hydrolysis of lignocellulosic biomass, this review exposed knowledge gaps that should be covered in future work in order to fully understand how enzyme synergism works: e.g. elucidating protein to protein interactions that exist between these enzymes in establishing synergy; and the effect of lignocellulose degradation products of one enzyme on the behaviour of the other enzyme and ultimately their synergistic relationship.