Purification and properties of β-N-acetylhexosaminidase from the mollusc Helicella ericetorum Müller

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Abstract
A .beta.-N-acetylhexosaminidase [EC 3.2.1.52] was purified 330-fold from the digestive gland of the terrestrial mollusk H. ericetorum Mueller. Its pH optimum was 4.5 for both .beta.-N-acetylglucosaminidase and .beta.-N-acetylgalactosaminidase activities in 2 buffer solutions; it was fully stable at 37.degree. C for 2 h in the pH range 3.8-4.6 and showed 1 isoelectric point (pH 4.83). The estimated MW was 120,000-145,000. The enzyme showed an endo-.beta.-N-acetylhexosaminidase activity on natural substrates such as ovalbumin, ovomucoid, chondroitin 4-sulfate, chitin and hyaluronic acid. Two forms of the enzyme were separated by preparative polyacrylamide-gel electrophoresis. Km and Vmax. for p-nitrophenyl 2-acetamido-2-deoxy-.beta.-D-glucopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-.beta.-D-galactopyranoside were 0.43 mM, 30.1 .mu.mol of p-nitrophenol/min per mg and 0.19 mM, and 8.6 .mu.mol of p-nitrophenol/min per mg, respectively. It was inhibited by Hg2+, Fe3+, acetate, some lactones, N-acetylgalactosamine, N-acetylglucosamine and mannose. Mixed-substrates analysis and Ki values for competitive inhibitors indicated that .beta.-N-acetylglucosaminidase and .beta.-N-acetylgalactosaminidase activities were catalyzed by the enzyme at the same active site.