Functional expression and characterization of an endo-1,4-β-mannosidase from Triticum aestivum in Pichia pastoris

Abstract

Endo-1,4-β-mannosidases catalyze the cleavage of the β-d-1,4-mannopyranosyl linkage in mannan and have potential biotechnological applications in biofuel production, manno-oligosaccharide production, cleansing, and food and feed industries. In this study, an endo-1,4-β-mannosidase gene (TaMan3A) from wheat (Triticum aestivum) was successfully expressed in Pichia pastoris, and the antifungal effectiveness and manno-oligosaccharide production of the enzyme were evaluated. The purified TaMan3A exhibited a molecular weight of approximately 43 kDa, and its highest enzyme activity at pH 4.0 and 40 °C. Comparisons with other β-mannanases showed that TaMan3A had a conserved mannan-binding V-shaped groove, catalytic acid/base residue (E179), and nucleophilic residue (E297). Antifungal assays for TaMan3A against seven fungi commonly associated with wheat kernels showed that this enzyme had higher inhibitory effects on hyphal growth of the field fungi Fusarium graminearum and Alternaria sp. in comparison with that of storage fungi. TaMan3A could hydrolyze mannan polymers of galactomannan and Konjac glucomannan to mannobiose, mannotriose, and mannotetraose as the main products. Overall, these results showed the potential of TaMan3A for enhancing host resistance against fungal pathogens in wheat and manno-oligosaccharide production in the feed and food industries.