Purification, biochemical and biophysical characterization of an acidic α-galactosidase from the seeds of Annona squamosa (custard apple)

Alpha galactosidase is an exoglycosidase that cleaves alpha-D-galactose and has numerous applications in medicine, biotechnology, food and pharma industries. In this study, a low molecular weight acidic alpha-galactosidase was identified from the seeds of custard apple. The purification of alpha-galactosidase from the crude extract of defatted seeds was achieved by employing ammonium sulphate fractionation, hydrophobic interaction and gel filtration chromatographic techniques. The purified custard apple alpha-galactosidase (CaG) migrated as a single band in native PAGE corresponding tomolecular weight of similar to 67 kDa and cleaved chromogenic, fluorogenic and natural substrates. CaGwas found to be a heterodimer with subunit masses of 40 and 30 kDa. The kinetic parameters such as KM and Vmax were found to be 0.67 mMand 1.5 U/mg respectively with p-nitrophenyl alpha-D-galactopyranoside. Galactose, methyl alpha-D-galactopyranoside and D-galacturonic acid inhibited CaG activity inmixed mode. The CD spectral analysis at far UV region showed that purified CaG exists predominantly as helix (35%), beta sheets (16.3%) and random coils (32.3%) in its secondary structure. These biochemical and biophysical properties of CaG provide leads to understand its primary sequence and glycan structures which will eventually define its novel physiological roles in plants and potential industrial applications. (C) 2021 Published by Elsevier B.V.