Hemicellulose is the second most abundant polysaccharide found in nature, and it is usually associated with cellulose and lignin in plant cell walls. The ?- mannans together with xylans are the major components of hemicelluloses. Mannanolytic enzymes are important reagents in industrial biorefinery processes such as the production of second generation biofuels from plant biomass. Many filamentous fungi produce ?-mannans degrading ?-1,4-mannanases that belong to the glycoside hydrolase 5 (GH5) and GH26 families. A. nidulans grown with sugars and ?-mannans as sole carbon source predominantly secreted an endo-?-1,4-mannanases (Man5C) and a hypothetical protein (HP). We investigated the enzymatic functions of a secreted HP of which the production was induced by ?-mannans. Recombinant HP was prepared, and the reaction products from galactose-free ?-mannan generated by the purified enzyme product were analyzed using MALDI-TOF-MS, HPLC and TLC. Recombinant HP released mannobiose (M2), mannotriose (M3), and mannotetraose (M4) but not mannopentaose (M5) or higher manno-oligosaccharides when galactose-free ?-mannan was the substrate. HP had high catalytic efficiency (kcat/Km) toward mannohexaose (M6) compared with the endo-?-1,4-mannanase Man5C and notably converted M6 to M2, M3, and M4, with M3 being the predominant reaction product. These results indicated that HP is a ?-1,4-mannanase (Man134A) that belongs to a new glycoside hydrolase family 134 (GH134) in the Carbohydrate-Active enZYmes (CAZy) classification. The growth phenotype of a man134A disruptant was poor when ?-mannans were the sole carbon source, indicating that Man134A is involved in ?-mannan degradation in vivo. These findings indicate a hitherto undiscovered mechanism of ?-mannan degradation that is enhanced by ?-1,4-mannanase, Man134A, when combined with other mannanolytic enzymes including various ?-1,4-mannanases belonging to the GH5 and GH26.
Full conference title:
- Fungal Genetics Conference 30th (2019)