Sripuan Thida, Aoki Kazuhiro, Yamamoto Kenji, Tongkao Dararat, Kumagai Hidehiko
Department of Chemistry, Faculty of Science, Chiang Mai University, Thailand.
Biosci Biotechnol Biochem. 2003 Jul;67(7):1485-91. doi: 10.1271/bbb.67.1485.
Alpha-galactosidase was purified from a fresh fruiting body of Ganoderma lucidum by precipitation with ammonium sulfate and column chromatographies with DEAE-Sephadex and Con A-Sepharose. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis. Its N-terminal amino acid sequence was similar to that of Mortierella vinacea alpha-galactosidase. The molecular mass of the enzyme was about 56 kDa by SDS-polyacrylamide gel electrophoresis, and about 249 kDa by gel filtration column chromatography. The optimum pH and temperature were 6.0 and 70 degrees C, respectively. The enzyme was fully stable to heating at 70 degrees C for 30 min. It hydrolyzed p-nitrophenyl-alpha-D-galactopyranoside (Km=0.4 mM) but hydrolyzed little o-nitrophenyl-alpha-D-galactopyranoside. It also hydrolyzed melibiose, raffinose, and stachyose. The enzyme catalyzed the transgalactosylation reaction which synthesized melibiose. The product was confirmed by various analyses.
通过硫酸铵沉淀以及用DEAE-葡聚糖凝胶和刀豆球蛋白A-琼脂糖凝胶进行柱色谱法，从新鲜的灵芝子实体中纯化出α-半乳糖苷酶。纯化后的酶在聚丙烯酰胺凝胶电泳上呈现均一性。其N端氨基酸序列与葡酒毛霉α-半乳糖苷酶的序列相似。通过SDS-聚丙烯酰胺凝胶电泳测定该酶的分子量约为56 kDa，通过凝胶过滤柱色谱法测定约为249 kDa。最适pH和温度分别为6.0和70℃。该酶在70℃加热30分钟时完全稳定。它能水解对硝基苯基-α-D-吡喃半乳糖苷（Km = 0.4 mM），但对邻硝基苯基-α-D-吡喃半乳糖苷几乎不水解。它还能水解蜜二糖、棉子糖和水苏糖。该酶催化合成蜜二糖的转半乳糖基化反应。产物通过各种分析得以确认。
