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

Samkelo Malgas; Mariska Thoresen; Vuyani Moses; Earl Prinsloo; J Susan van Dyk; Brett I Pletschke

doi:10.1016/j.csbj.2022.06.038

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

Comput Struct Biotechnol J. 2022 Jun 17:20:3140-3150. doi: 10.1016/j.csbj.2022.06.038. eCollection 2022.

Authors

Samkelo Malgas^{1

2}, Mariska Thoresen¹, Vuyani Moses³, Earl Prinsloo⁴, J Susan van Dyk⁵, Brett I Pletschke¹

Affiliations

¹ Enzyme Science Programme (ESP), Department of Biochemistry and Microbiology, Rhodes University, Makhanda, Eastern Cape 6140, South Africa.
² Department of Biochemistry, Genetics and Microbiology, University of Pretoria, Hatfield, Gauteng 0028, South Africa.
³ Research Unit in Bioinformatics (RUBi), Department of Biochemistry and Microbiology, Rhodes University, Makhanda, Eastern Cape 6140, South Africa.
⁴ Biotechnology Innovation Centre, Rhodes University, Makhanda, Eastern Cape 6140, South Africa.
⁵ Forest Products Biotechnology, University of British Columbia, 2424 Main Mall, Vancouver, British Columbia V6T1Z4, Canada.

Abstract

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.

Keywords: Galactomannan; Non-catalytic binding; β-mannosidase.