Koga D, Hoshika H, Matsushita M, Tanaka A, Ide A, Kono M
Laboratory of Biochemistry, Yamaguchi University, Yoshida, Japan.
Biosci Biotechnol Biochem. 1996 Feb;60(2):194-9. doi: 10.1271/bbb.60.194.
Beta-N-Acetylhexosaminidase (EC 3.2.1.52) was purified from the liver of a prawn, Penaeus japonicus, by ammonium sulfate fractionation and chromatography with Sephadex G-100, hydroxylapatite, DEAE-Cellulofine, and Cellulofine GCL-2000-m. The purified enzyme showed a single band keeping the potential activity on both native PAGE and SDS-PAGE. The apparent molecular weight was 64,000 and 110,000 by SDS-PAGE and gel filtration, respectively. The pI was less than 3.2 by chromatofocusing. The amino-terminal amino acid sequence was NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly- Glu-Pro-. The optimum pH and temperature were 5.0 to 5.5 and 50 degrees C, respectively. The enzyme was stable from pH 4 to 11, and below 55 degrees C. It was 39% inhibited by 10 mM HgCl2. Steady-state kinetic analysis was done with the purified enzyme using N-acetylchitooligosaccharides (GlcNAcn, n = 2 to 6) and p-nitrophenyl N-acetylchitooligosaccharides (pNp-beta-GlcNAcn, n = 1 to 3) as the substrates. The enzyme hydrolyzed all of these substrates to release monomeric GlcNAc from the non-reducing end of the substrate. The parameters of Km and kcat at 25 degrees C and pH 5.5 were 0.137 mM and 598 s-1 for pNp-beta-GlcNAc, 0.117 mM and 298 s-1 for GlcNAc2, 0.055 mM and 96.4 s-1 for GlcNAc3, 0.044 mM and 30.1 s-1 for GlcNAc4, 0.045 mM and 14.7 s-1 for GlcNAc5, and 0.047 mM and 8.3 s-1 for GlcNAc6, respectively. These results suggest that this beta-N-acetylhexosaminidase is an exo-type hydrolytic enzyme involved in chitin degradation, and prefers the shorter substrates.
β-N-乙酰己糖胺酶（EC 3.2.1.52）通过硫酸铵分级分离及使用葡聚糖G-100、羟基磷灰石、DEAE-纤维素和纤维素GCL-2000-m进行色谱法，从日本对虾的肝脏中纯化得到。纯化后的酶在天然聚丙烯酰胺凝胶电泳（Native PAGE）和十二烷基硫酸钠聚丙烯酰胺凝胶电泳（SDS-PAGE）上均显示出单一活性条带。通过SDS-PAGE和凝胶过滤法测得的表观分子量分别为64,000和110,000。通过聚焦色谱法测得其等电点小于3.2。氨基末端氨基酸序列为NH2-Thr-Leu-Pro-Pro-Pro-Trp-Gly-Trp-Ala-?-Asp-Gln-Gly-Val-?-Val-Lys-Gly-Glu-Pro-。最佳pH值和温度分别为5.0至5.5以及50℃。该酶在pH值4至11且温度低于55℃时稳定。10 mM HgCl2对其有39%的抑制作用。使用N-乙酰几丁寡糖（GlcNAcn，n = 2至6）和对硝基苯基N-乙酰几丁寡糖（pNp-β-GlcNAcn，n = 1至3）作为底物，对纯化后的酶进行稳态动力学分析。该酶可水解所有这些底物，从底物的非还原端释放出单体GlcNAc。在25℃和pH 5.5条件下，pNp-β-GlcNAc的Km和kcat参数分别为0.137 mM和598 s-1，GlcNAc2的为0.117 mM和298 s-1，GlcNAc3的为0.055 mM和96.4 s-1，GlcNAc4的为0.044 mM和30.1 s-1，GlcNAc5的为0.045 mM和14.7 s-1，GlcNAc6的为0.047 mM和8.3 s-1。这些结果表明，这种β-N-乙酰己糖胺酶是一种参与几丁质降解的外切型水解酶，且更倾向于较短的底物。
