title: Purification and characterization of a class II α-Mannosidase from Moringa oleifera seed kernels.
abstract: α-Mannosidase (EC. 3.2.1.114) belonging to class II glycosyl hydrolase family 38 was purified from Moringa oleifera seeds to apparent homogeneity by conventional protein purification methods followed by affinity chromatography on Con A Sepharose and size exclusion chromatography. The purified enzyme is a glycoprotein with 9.3 % carbohydrate and exhibited a native molecular mass of 240 kDa, comprising two heterogeneous subunits with molecular masses of 66 kDa (α-larger subunit) and 55 kDa (β-smaller subunit). Among both the subunits only larger subunit stained for carbohydrate with periodic acid Schiff's staining. The optimum temperature and pH for purified enzyme was 50 °C and pH 5.0, respectively. The enzyme was stable within the pH range of 3.0-7.0. The enzyme was inhibited by EDTA, Hg(2+), Ag(2+), and Cu(2+). The activity lost by EDTA was completely regained by addition of Zn(2+). The purified enzyme was characterized in terms of the kinetic parameters K m (1.6 mM) and V max (2.2 U/mg) using para-nitrophenyl-α-D-mannopyranoside as substrate. The enzyme was very strongly inhibited by swainsonine (SW) at 1 μM concentration a class II α-Mannosidase inhibitor, but not by deoxymannojirimycin (DMNJ). Chemical modification studies revealed involvement of tryptophan at active site. The inhibition by SW and requirement of the Zn(2+) as a metal ion suggested that the enzyme belongs to class II α-Mannosidase.
authors.affiliation: Department of Biochemistry, Central University of Rajasthan, Kishangarh, Ajmer, 305817, Rajasthan, India, kirankumar@curaj.ac.in.
authors.forename: Kiran Kumar
authors.initials: KK
authors.lastname: Tejavath
authors.forename: Siva Kumar
authors.initials: SK
authors.lastname: Nadimpalli
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods; D008970:Molecular Weight; D029588:Moringa oleifera / Q000196:embryology / Q000201:enzymology*; D012639:Seeds / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D043323:alpha-Mannosidase
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