James J A, Berger J L, Lee B H
Department of Food Science and Agricultural Chemistry, Macdonald Campus of McGill University, 21111 Lakeshore, Ste-Anne de Bellevue, Québec, H9X 3V9, Canada.
Curr Microbiol. 1997 Mar;34(3):186-91. doi: 10.1007/s002849900166.
An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein liquid chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-beta-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I2/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45 degrees C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases.
利用配备Mono Q离子交换器和两个串联的Superose 12凝胶过滤柱的快速蛋白质液相色谱系统（FPLC），从嗜酸乳杆菌中纯化出一种细胞内葡糖淀粉酶（E.C. 3.2.1.3），使其达到同质状态。使用特异性显色底物对硝基苯基-β-麦芽糖苷对酶活性进行定量。然后用制备型凝胶电泳进一步纯化活性酶组分。纯化酶的非变性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）显示出一条分子量为47 kDa的单一蛋白条带。通过纯化蛋白在经I2/KI染色的0.025%淀粉-聚丙烯酰胺凝胶上降解淀粉的能力，证实了其葡糖淀粉酶活性。葡糖淀粉酶在pH 6.0和45℃时表现出最佳催化活性，该酶的等电点接近4.39。该葡糖淀粉酶含有高水平的亲水性氨基酸，与真菌葡糖淀粉酶相当。
