Purification and characterization of beta-N-acetyl-D-hexosaminidase from the mid-gut gland of scallop (Patinopecten yessoensis).
beta-N-Acetyl-D-hexosaminidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified by making an acetone-dried preparation of the mid-gut gland, extracting with 50 mM citrate-phosphate buffer (pH 4.0) (about 13% of the extracted proteins was beta-N-acetyl-D-hexosaminidase), ammonium sulfate fractionation, and column chromatographies on CM-Sepharose and DEAE-Sepharose. The purified beta-N-acetyl-D-hexosaminidase was homogeneous on SDS-PAGE, and sufficiently free from other exo-type glycosidases. The molecular weight was 56,000 by SDS-PAGE. The enzyme hydrolyzed both p-nitrophenyl beta-N-acetyl-D-glucosaminide and p-nitrophenyl beta-N-acetyl-D-galactosaminide. For p-nitrophenyl beta-N-acetyl-D-glucosaminide, the pH optimum was 3.7, the optimum temperature was 45 degrees C, and the Km was 0.24 mM. For p-nitrophenyl beta-N-acetyl-D-galactosaminide, these were pH 3.4, 45 degrees C, and 0.15 mM, respectively. The enzyme liberated non-reducing terminal beta-linked N-acetyl-D-glucosamine or N-acetyl-D-galactosamine from various 2-aminopyridyl derivatives of oligosaccharides of N-glycan or glycolipid type except of GM2-tetrasaccharide. As the enzyme was stable around pH 3.5-5.5, it may be useful for long time reactions around the optimum pH.
Sakai
T
T
Research Institute for Glycotechnology Co., Ltd., Aomori, Japan.
Nakanishi
Y
Y
Kato
I
I
D000818:Animals; D002240:Carbohydrate Sequence; D004064:Digestive System / Q000201:enzymology; D004790:Enzyme Induction; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008974:Mollusca / Q000201:enzymology*; D009844:Oligosaccharides / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D001619:beta-N-Acetylhexosaminidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
D009844:Oligosaccharides; D001619:beta-N-Acetylhexosaminidases