Production, purification and characterization of a constitutive intracellular ?-galactosidase from the thermophilic fungus Humicola sp

The thermophilic fungus Humicola sp constitutively produces intracellular alpha-galactosidase (1.33 U mg-1 protein) within 48 h at 45 degrees C in shaken flasks, when grown in a medium containing 7% wheat bran extract as a carbon source and 0.5% yeast extract as a nitrogen source. The enzyme has been purified to homogeneity by ultrafiltration, ethanol precipitation, DEAE cellulose and Sephacryl S-300 chromatography with a 124-fold increase in specific activity and 29.5% recovery. The molecular weight of the enzyme is 371.5 kDa by gel filtration on Sephacryl S-300 and 87.1 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme has an optimum temperature of 65 degrees C and an optimum pH of 5.0. Humicola alpha-galactosidase is a glycoprotein with 8.3% carbohydrate content and is acidic in nature with a pI of 4.0. The KmS for p-nitrophenyl-alpha-D-galactopyranoside, O-nitrophenyl-alpha-D-galactopyranoside, raffinose and stachyose are 0.279, 0.40, 1.45 and 1.42 mM respectively. The enzyme activity was strongly inhibited by Ag+ and Hg2+. D-Galactose inhibited alpha-galactosidase competitively and the inhibition constant (Ki) for galactose was 11 mM.