Title
Purification, biochemical and biophysical characterization of a zinc dependent α-mannosidase isoform III from Custard Apple (Annona squamosa) seeds
Author
Kavyashree Sakharayapatna Ranganatha, Lipsa Sahoo, Ashapogu Venugopal, Siva Kumar Nadimpalli
Year
2019
Journal
International Journal of Biological Macromolecules
Abstract
In the present study, out of three isoforms of α-mannosidase identified in the crude extract of defatted Custard apple seed powder, isoform III has been purified to homogeneity by two-step chromatography: hydrophobic interaction and gel filtration. The purified Custard apple α-mannosidase isoform III (CAM) hydrolyzed both chromogenic (p-nitrophenyl-α-D-mannopyranoside) and fluorescent (4-methylumbelliferyl α-D-mannopyranoside) substrates. Custard apple α-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 ~ 75, 68, 56 and 50 kDa respectively). Temperature and pH optima were found to be 50 °C and 4.0–5.0 respectively and CAM was stable up to 60–70 °C. The enzymatic activity of CAM was inhibited by EDTA, Ag+, Hg2+, Ni2+ and swainsonine (IC50 value of 1.5 μM). CAM was observed to be a metallo enzyme requiring zinc for its activity. Kinetic parameters KM 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%), β 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.
Instrument
J-1500
Keywords
Circular dichroism, Secondary structure, Thermal stability, Biochemistry