Purification and characterization of alpha-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular alpha -galactosidase was purified to electrophoretic homog eneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the e nzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl alpha -D- galactosides but efficiently hydrolyzed alpha -glycosidically linked non-re ducing terminal galactopyranosyl residues occurring in natural substrates s uch as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of pol ymeric galactomannans leading to precipitation of the polymers. Stereochemi cal course of hydrolysis of two substrates, 4-nitrophenyl alpha -galactopyr anoside and galactosyl(1)mannotriose, followed by H-1 NMR spectroscopy, poi nted out the alpha -anomer of D-galactose was the primary product of hydrol ysis from which the beta -anomer was formed by mutarotation. Hence the enzy me is a retaining glycosyl hydrolase. In accord with its retaining characte r the enzyme catalyzed transgalactosylation from 4-nitrophenyl alpha -galac topyranoside as a glycosyl donor. Amino acid sequence alignment of N-termin al and two internal sequences suggested that the enzyme is a member of fami ly 27 of glycosyl hydrolases. (C) 2000 Elsevier Science B.V. All rights res erved.