Studies on the Glycosidases of Jack Bean Meal: III. CRYSTALLIZATION AND PROPERTIES OF β-N-ACETYLHEXOSAMINIDASE

Abstract

Abstract A method has been devised for the isolation of β-N-acetyl-hexosaminidase from jack bean meal in electrophoretically homogeneous and crystalline form. The procedure involves ammonium sulfate and alcohol fractionation followed by Sephadex G-200 filtration and chromatography on columns of DEAE-Sephadex and carboxymethyl-Sephadex. The molecular weight of this enzyme is estimated to be about 100,000 by gel filtration. The crystalline enzyme hydrolyzes both p-nitrophenyl β-2-acetamido-2-deoxy-d-glucopyranoside and p-nitrophenyl β-2-acetamido-2-deoxy-d-galactopyranoside. For p-nitrophenyl β-2-acetamido-2-deoxy-dglucopyranoside, the pH optimum is between 5.0 and 6.0, Km is 0.64 mm and Vmax is 137.2 µmoles per min per mg. For p-nitrophenyl β-2-acetamido-2-deoxy-d-galactopyranoside, the pH optimum is between 3.5 and 4.0, Km is 0.31 mm and Vmax is 70.9 µmoles per min per mg. Studies of pH and thermal inactivation, mixed-substrate analysis, and the Ki values for several competitive inhibitors indicate that the β-N-acetylglucosaminidase and the β-N-acetylgalactosaminidase activities are catalyzed by the same enzyme at the same site. The crystalline enzyme liberates β-linked terminal N-acetylglucosamine and N-acetylgalactosamine from various natural and synthetic substrates.