Class II α-mannosidase from Trichosanthes anguina(Snake Gourd) seeds: Purification and biochemical characterization

Abstract

Plant α-mannosidase is an exoglycosidase, which plays an important role in the removal of terminal α (1–2, 3, 6) linked mannosyl residues from the non-reducing ends of oligosaccharides or N-glycans of glycoproteins. In the present study, a class II α-mannosidase has been purified to homogeneity from the defatted seed extracts of an edible non-legume vegetable, Snake gourd (Trichosanthes anguina), by a combination of hydrophobic interaction, anion exchange and gel filtration chromatography techniques. The purified enzyme migrated as a single band in native PAGE under non-reducing and non-denaturing conditions. The molecular mass of purified enzyme was estimated to be ~235 kDa by gel filtration chromatography. In SDS-PAGE, under reducing conditions, purified α-mannosidase resolved in to four subunits with an approximate molecular masses of 100, 75, 63 and 48 kDa respectively and showed reactivity with an antiserum raised against jack bean α-mannosidase. The authenticity of the purified enzyme was confirmed by zymogram analysis with 4-methylumbelliferyl α-D-mannopyranoside. The pH and temperature optima of mannosidase were found to be 4.0–5.0 and 40 °C respectively and the enzyme was thermally stable up to 60 °C. The Kinetic parameters, K M and Vmax of the enzyme estimated with p-nitrophenyl α-D-mannopyranoside were 0.966 mM and 0.1009 μmol-1 min-1 mL-1 respectively. T. anguina mannosidase belongs to class II family of mannosidases as demonstrated by the inhibition studies with swainsonine, with IC 50 value ranging from 500 to 750 nM. The rate of hydrolysis of p-nitrophenyl α-D-mannopyranoside by snake gourd α-mannosidase was inhibited noncompetitively by swainsonine and methyl-α-D-mannopyranoside; mixed mode by mannose and competitively inhibited by D-glucosamine and D-mannosamine.