Biochemical analyses of a novel acidophilic GH5 -mannanase from Trichoderma asperellum ND-1 and its application in mannooligosaccharides production from galactomannans

In this study, an acidophilic GH5 beta-mannanase (TaMan5) from Trichoderma asperellum ND-1 was efficiently expressed in Pichia pastoris (a 2.0-fold increase, 67.5 +/- 1.95 U/mL). TaMan5 displayed the highest specificity toward locust bean gum (K-m = 1.34 mg/mL, V-max = 749.14 mu mol/min/mg) at pH 4.0 and 65 degrees C. Furthermore, TaMan5 displayed remarkable tolerance to acidic environments, retaining over 80% of its original activity at pH 3.0-5.0. The activity of TaMan5 was remarkably decreased by Cu2+, Mn2+, and SDS, while Fe2+/Fe3+ improved the enzyme activity. A thin-layer chromatography (TLC) analysis of the action model showed that TaMan5 could rapidly degrademannan/MOS intomannobiose without mannose via hydrolysis action as well as transglycosylation. Site-directed mutagenesis results suggested that Glu(205), Glu(313), and Asp(357) of TaMan5 are crucial catalytic residues, with Asp(152) playing an auxiliary function. Additionally, TaMan5 and commercial alpha-galactosidase displayed a remarkable synergistic effect on the degradation of galactomannans. This study provided a novel beta-mannanase with ideal characteristics and can be considered a potential candidate for the production of bioactive polysaccharide mannobiose.