Sakai T, Nakanishi Y, Kato I
Research Institute for Glycotechnology Co., Ltd., Aomori, Japan.
Biosci Biotechnol Biochem. 1993 Jun;57(6):965-8. doi: 10.1271/bbb.57.965.
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.
β-N-乙酰-D-己糖胺酶是从虾夷扇贝的中肠腺中分离出来的。通过对中肠腺进行丙酮干燥制备、用50 mM柠檬酸-磷酸盐缓冲液（pH 4.0）提取（提取的蛋白质中约13%是β-N-乙酰-D-己糖胺酶）、硫酸铵分级分离以及在CM-琼脂糖和DEAE-琼脂糖上进行柱色谱法来纯化该酶。纯化后的β-N-乙酰-D-己糖胺酶在SDS-PAGE上呈均一性，且充分不含其他外切型糖苷酶。通过SDS-PAGE测定其分子量为56,000。该酶可水解对硝基苯基β-N-乙酰-D-葡糖胺和对硝基苯基β-N-乙酰-D-半乳糖胺。对于对硝基苯基β-N-乙酰-D-葡糖胺，最适pH为3.7，最适温度为45℃，Km为0.24 mM。对于对硝基苯基β-N-乙酰-D-半乳糖胺，这些参数分别为pH 3.4、45℃和0.15 mM。该酶能从N-聚糖或糖脂型寡糖的各种2-氨基吡啶基衍生物中释放出非还原末端β-连接的N-乙酰-D-葡糖胺或N-乙酰-D-半乳糖胺，但GM2-四糖除外。由于该酶在pH 3.5 - 5.5左右稳定，它可能适用于在最适pH附近进行长时间反应。
