Purification and characterization of guar galactomannan degrading alpha-galactosidase from Aspergillus oryzae DR-5

Abstract

alpha-Galactosidase from A. oryzae DR-5 was induced in the presence of melibiose, raffinose, galactose, and locust bean galactomannan. The enzyme was purified to homogeneity by precipitation with acetone followed by ion-exchange chromatography using DEAE-Sephacel. The purified enzyme showed a single band in both nondenaturing-PAGE and SDS-PAGE. The enzyme was a glycoprotein in nature by activity staining. The molecular weight of the purified enzyme was 93- 95 kDa by SDS-PAGE. The enzyme exhibited the optimum pH and temperature at 4.7 and 60degreesC, respectively. alpha-Galactosidase activity was strongly inhibited by Ag2+, Hg2+, Cu2+, and galactose. EDTA, 1,10-phenanthraline, and PMSF did not inhibit the enzyme activity, whereas N-bromosuccinimide completely inhibited enzyme activity. Investigation by TLC showed complete hydrolysis of stachyose and raffinose in soymilk in 3 h at pH 5.0 and 50degreesC.