Okamura M
Laboratory of Biochemistry and Nutrition, Kanagawa Prefectural College of Nursing and Medical Technology, Yokohama, Japan.
J Nutr Sci Vitaminol (Tokyo). 2001 Jun;47(3):258-62. doi: 10.3177/jnsv.47.258.
L-Gulono-1,4-lactone oxidase activity was detected in G. frondosa: therefore its properties were studied after purification. A 766-fold purified preparation of the enzyme from fresh fruit bodies was obtained by means of a seven-step procedure, the overall yield being 14%. The purified enzyme gave a single band on polyacrylamide gel electrophoresis and its absorption spectrum exhibited the characteristic of a flavoenzyme. The enzyme produced L-ascorbic acid and H2O2, with L-gulono-1,4-lactone (GL) as the substrate and oxygen as the electron acceptor, and was optimally active at around pH 7.0 and 45 degrees C. Its molecular mass was determined to be 250 kDa on gel filtration, while the dissociated enzyme exhibited a molecular mass of 69 kDa on SDS-polyacrylamide gel electrophoresis, but the true molecular weight is unknown because of the trypsin treatment in the purification process. The apparent Km value for GL was 24+/-1 mM. Its substrate specificity was extremely high and, assuming that for GL to be 100, the following results were obtained: D-mannono-, 25: D-glucono-, 4; L-idono-, 3; L-galactono-1,4-lactone, 2; and 15 other lactones tested, 0. It is presumed that this enzyme is similar to animal GL-oxidase, ascomycetes D-arabinonolactone oxidase, etc.
在灰树花中检测到了L-古洛糖酸-1,4-内酯氧化酶活性：因此在纯化后对其性质进行了研究。通过七步程序从新鲜子实体中获得了该酶的766倍纯化制剂，总产率为14%。纯化后的酶在聚丙烯酰胺凝胶电泳上呈现单一谱带，其吸收光谱显示出黄素酶的特征。该酶以L-古洛糖酸-1,4-内酯（GL）为底物、氧气为电子受体产生L-抗坏血酸和H2O2，在pH 7.0左右和45℃时活性最佳。通过凝胶过滤测定其分子量为250 kDa，而解离后的酶在SDS-聚丙烯酰胺凝胶电泳上显示分子量为69 kDa，但由于纯化过程中的胰蛋白酶处理，其真实分子量未知。GL的表观Km值为24±1 mM。其底物特异性极高，假设GL的底物特异性为100，则得到以下结果：D-甘露糖酸-，25；D-葡萄糖酸-，4；L-艾杜糖酸-，3；L-半乳糖酸-1,4-内酯，2；以及测试的其他15种内酯，0。据推测，这种酶类似于动物GL-氧化酶、子囊菌D-阿拉伯糖内酯氧化酶等。
