Purification and characterization of a beta-N-acetylhexosaminidase of sea-squirt.

A beta-N-acetylhexosaminidase [EC 3.2.1.30] was isolated from internal organs of the sea-squirt, Styela plicata. The enzyme was purified 1,560-fold in 5% yield. The preparation was fairly homogeneous as examined by disc and SDS polyacrylamide gel electrophoresis and Sephadex G-200 chromatography. The molecular weight of the enzyme was estimated to be 132,000 by gel chromatography and 66,000 by SDS polyacrylamide gel electrophoresis. Therefore, this beta-N-acetylhexosaminidase was considered to be a dimer. The optimum pH for activity was 4.0 but the enzyme was stable in the pH range from 5 to 6. The isoelectric point was 4.99. This enzyme was inhibited by Fe2+, Hg2+, Ag+, and PCMB but not by acetate. The isolated enzyme hydrolyzed both p-nitrophenyl-N-acetyl-beta-D-glucosaminide and p-nitrophenyl-N-acetyl-beta-D-galactosaminide. The hydrolysis rate of p-nitrophenyl-N-acetyl-beta-D-galactosaminide was 43% of that of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The enzyme liberated N-acetylhexosamine from asialodegalactosyl ovomucoid glycopeptide, asialodegalactosyl fetuin glycopeptide and the fragment of hyaluronic acid prepared by hyaluronidase treatment.