Purification, biochemical and biophysical characterization of a zinc dependent α-mannosidase isoform III from Custard Apple (Annona squamosa) seeds

In the present study, out of three isoforms of alpha-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform Ill has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple alpha-mannosidase isoform (CAM) hydrolyzed both chromogenic (p-nitrophenyl-alpha-D-mannopyranoside) and fluorescent (4-methylumbelliferyl alpha-D-mannopyranoside) substrates. Custard apple alpha-mannosidase migrated as a single band in native PAGE, showed about 220 kDa molecular mass in gel filtration and in SDS PAGE, dissociated into four bands (Mr similar to 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 degrees C and 4.0-5.0 respectively and CAM was stable up to 60-70 degrees C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC50 value of 1.5 mu M). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters K-M and Vmax were found to be 1.75 mM and 0.068 U/mL respectively. The CD spectral analysis at far UV region (190-300 nm) shows that purified CAM exists as helix (30.4%), beta turns (18%) and random coils (29.7%) in its secondary structure. Chemical modification studies with N-Bromosuccinimide revealed the presence of tryptophan in its active site. (C) 2019 Elsevier B.V. All rights reserved.