alpha-N-Acetylgalactosaminidase (alpha-GalNAc-ase; EC.3.2.1.49) is an exoglycosidase specific for the hydrolysis of terminal alpha-linked N-acetylgalactosamine in various sugar chains. The cDNA corresponding to the alpha-GalNAc-ase gene was cloned from Aspergillus niger, sequenced, and expressed in the yeast Saccharomyces cerevisiae. The alpha-GalNAc-ase gene contains an open reading frame which encodes a protein of 487 amino acid residues. The molecular mass of the mature protein deduced from the amino acid sequence of this reading frame is 54 kDa. The recombinant protein was purified to apparent homogeneity and biochemically characterized (pI 4.4. K-M 0.56 mmol/l for 2-nitrophenyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, and optimum enzyme activity was achieved at pH 2.0-2.4 and 50-55 degrees C). Its molecular weight was determined by analytical ultracentrifuge measurement and dynamic light scattering. Our experiments confirmed that the recombinant alpha-GalNAc-ase exists as two distinct species (70 and 130 kDa) compared to its native form, which is purely monomeric. N-Glycosylation was confirmed at six of the eight potential N-glycosylation sites in both wild type and recombinant alpha-GalNAc-ase. (C) 2011 Elsevier Inc. All rights reserved.