The multiple alpha-galactosidases from Streptomyces griseoloalbus-alpha-Gal I, alpha-Gal II and alpha-Gal III were purified to homogeneity by a two-step chromatographic process. The molecular masses and pl of the three enzymes were 72, 57 and 35 kDa, and 4.41, 5.6 and 6.13, respectively. alpha-Gal I showed N-terminal sequence homology to S. coelicolor A3(2) family 27 alpha -galactosidase. The optimum pH and temperature of the three a-galactosidases were 5.0, 6.5 and 5.5 and 65, 50 and 55 C, respectively. alpha-Gal I was stable up to 65 degrees C and alpha-Gal II and alpha-Gal III up to 55 degrees C for 2 h. Based on the hydrolytic properties alpha-Gal I could be classified as a member of GH27 family and alpha-Gal II and alpha-Gal III as members of GH36 family. Metal cations like Hg(2+), Ag(2+) and Cu(2+) inhibited enzyme activity while Mg(2+) enhanced the activity of alpha-Gal I. Interestingly alpha-Gal I showed unusual tolerance to even higher concentrations of galactose, unlike the other two alpha-galactosidases, which were competitively inhibited by galactose. Melibiose was a competitive inhibitor of all three enzymes. Histidine, tryptophan and carboxylic residues were essential for catalytic action of the three alpha-galactosidases. (C) 2008 Elsevier Ltd. All rights reserved.