Purification and properties of ⍺-galactosidase from white-rot fungus Pleurotus florida

Abstract

⍺-Galactosidase was strongly induced in the white-rot fungus Pleurotus florida by arabinose than its natural substrates and was purified to homogeneity by acetone precipitation, ultrafiltration and DEAE-Sepharose chromatography. The enzyme was a monomeric protein with a molecular mass of ≅ 99 kDa, as revealed by native-PAGE and SDS-PAGE. ⍺-Galactosidase was optimally active at 55ºC for the hydrolysis of p-nitrophenyl-⍺-galactopyranoside (PNP⍺G) and lost its 20% and 50% of original activity in 30 min at 60ºC and 70ºC, respectively. The pH optimum of the enzyme was between 4.6 and 5.0. It was stable in a wide pH range (pH 4.0 to 9.0) at 55ºC for 2 h. The Ag⁺ and Hg²⁺ strongly inhibited the enzyme activity. Galactose, glucose, maltose and lactose also inhibited the enzyme activity, whereas N-bromosuccinimide treatment resulted in near total loss of acitivity. The Km and Vmax values of the enzyme for PNPαG were found to be 1.1 mM, and 77 μmol min⁻¹ mg⁻¹, respectively. ⍺-Galactosidase immobilized in agar was more effective for the degradation of raffinose than in the sodium alginate. TLC results indicated its potential for the removal of raffinose and stachyose in soymilk.