Biochemical analyses of a novel acidophilic GH5 β-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.

Automated summary

In this study, a acidophilic GH5 beta-mannanase with high activity and tolerance to acidic environments was efficiently expressed in Pichia pastoris. The enzyme showed specificity towards locust bean gum and exhibited optimal activity at pH 4.0 and 65 degrees C. Site-directed mutagenesis confirmed the crucial catalytic residues of the enzyme. Additionally, a synergistic effect was observed when the enzyme was combined with a commercial alpha-galactosidase.