Penicillium sp 23 alpha-galactosidase: purification and substrate specificity

alpha -Galactosidase, a glycoprotein with carbohydrate and protein in ratio 1:6, has been isolated from liquid culture of micromycete Penicillium sp. 23 and purified to homogeneous state by ammonium sulphate precipitation fol lowed by ion exchange and gel-filtration chromatography on TSK-gels. The Pe nicillium sp. 23 alpha -galactosidase specificity against a series of natur al and synthetic substrates has been studied. The enzyme was found to exhib it strict specificity towards the glycon and hydrolyze exclusively alpha -D -galactosides such as p-nitrophenyl-alpha -D-galactopyranoside (p-NPhGal), melibiose, raffinose and stachyose. The configuration at C1 and C4 atoms of substrate as well as substitution at C2 and C6 of substrate made an import ant contribution to the interaction with the enzyme. The tested alpha -gala ctosidase exerted the highest affinity (K-m) with respect to the synthetic substrate p-NPhGal and maximal rate of hydrolysis (V-max), about 10 times h igher, comparing with natural substrates (melibiose, raffinose and stachios e). The Penicillium sp. 23 alpha -galactosidase possesses wide specificity towards alpha -galactosidase hydrolysis link. type. splitting off at varyin g rates the terminal galactose from disaccharides, attached by alpha -1,2-, alpha -1,3- and alpha -1,6-links. The enzyme is ineffective towards disacc harides with alpha -1,4-link. The enzyme showed potential to splitting off alpha -1,3-bound terminal galactose residues from antigens of the human blo od group B(III) erythrocytes. (C) 2001 Elsevier Science Ltd. All rights res erved.