Purification and characterization of a blood group A2 degrading alpha-N-acetylgalactosaminidase from Clostridium perfringens

α-N-acetylgalactosaminidase is an exoglycosidase that can modify immunodominant carbohydrate epitopes on glycoproteins and glycolipids, by cleaving specific monosaccharide units from the nonreducing ends. α-N-acetylgalactosaminidase cleaves the terminal α-1,3 linked N-acetylgalactosamine residue from glycoconjugates specifically the blood type A epitope.; A highly purified preparation of α-N-acetylgalactosaminidase was obtained from Clostridium perfringens culture by salt precipitation, gel filtration, ion exchange chromatography, chromatofocusing, and high performance liquid chromatography. The final preparation was characterized for molecular weight, substrate specificity, pH optimum, and activity against the blood type A₂ epitope. The final preparation of Clostridium perfringens α-N-acetylgalactosaminidase was homogeneous by SDS-PAGE with a molecular mass of 72.1 kDa. The native molecular weight of the enzyme was determined as 69.05 kDa, by Sephacryl S-200 gel filtration. The enzyme was highly selective for terminal N-acetyl-α-D-galactosamine residues. There were no other significant glycosidase activities detected, specifically neuraminidase. The pH optimum of the enzyme was between 6.5 to 7.0 and activity was unaffected by ionic strength. No protease activity was detected and enzyme activity was stable at 4°C for twelve months. ELISA experiments demonstrated activity against blood type A₂ epitope, high tolerance of ionic strength up to 1600 mM NaCl, and stability in the pH range of 5.5 to 7.5 and at the temperature of 4°C, 24°C and 37°C. Importantly, the enzyme was active in red cell preservatives and plasma at pH 7.0. Therefore, this enzyme could be directly added to red blood cell units eliminating the need to lower the pH by extensive red cell washing as required by the current technology.; Clostridium perfringens α-N-acetylgalactosaminidase may be useful for enzymatic conversion of human blood group A₂ red cells to “universally transfusable” group O red cells. Enzymatic conversion of type A₂ red cell units could increase the type O blood supply from approximately 45 to 55% of all red cell units collected. Since it is difficult to obtain native enzyme in adequate yield, a recombinant Clostridium perfringens α-N-acetylgalactosaminidase expressed in high yields is potentially superior to eucaryotic native or recombinant lysozomal enzymes for applications in field of enzymatic conversion biotechnology.