来自短双歧杆菌K-110的α-L-阿拉伯吡喃糖苷酶和α-L-阿拉伯呋喃糖苷酶的纯化与特性研究，短双歧杆菌K-110是一种代谢人参皂苷Rb2和Rc的人体肠道厌氧菌。
Purification and characterization of alpha-L-arabinopyranosidase and alpha-L-arabinofuranosidase from Bifidobacterium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc.
作者信息
Shin Ho-Young, Park Sun-Young, Sung Jong Hwan, Kim Dong-Hyun
机构信息
College of Pharmacy, Kyung Hee University, Dongdaemun-ku, Seoul, Korea.
出版信息
Appl Environ Microbiol. 2003 Dec;69(12):7116-23. doi: 10.1128/AEM.69.12.7116-7123.2003.
Two arabinosidases, alpha-L-arabinopyranosidase (no EC number) and alpha-L-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. alpha-L-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 micro mol/min/mg. alpha-L-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 micro mol/min/mg. The molecular mass of alpha-L-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that of alpha-L-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. alpha-L-Arabinopyranosidase and alpha-L-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40 degrees C and pH 4.5 and 45 degrees C, respectively. Both purified enzymes were potently inhibited by Cu(2+) and p-chlormercuryphenylsulfonic acid. alpha-L-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinopyranoside, followed by ginsenoside Rb2. alpha-L-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-alpha-L-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-beta-galactopyranoside or p-nitrophenyl-beta-D-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified alpha-L-arabinosidases. This is the first reported purification of alpha-L-arabinopyranosidase from an anaerobic Bifidobacterium sp.
从人参皂苷代谢短双歧杆菌K - 110中纯化出两种阿拉伯糖苷酶，即α - L - 阿拉伯吡喃糖苷酶（无酶委员会编号）和α - L - 阿拉伯呋喃糖苷酶（酶委员会编号3.2.1.55），该菌株是从人类肠道微生物群中分离得到的。通过硫酸铵分级沉淀、DEAE - 纤维素、丁基Toyopearl、羟基磷灰石Ultrogel、QAE - 纤维素和Sephacryl S - 300 HR柱层析相结合的方法，将α - L - 阿拉伯吡喃糖苷酶纯化至表观均一，最终比活性为8.81微摩尔/分钟/毫克。通过硫酸铵分级沉淀、DEAE - 纤维素、丁基Toyopearl、羟基磷灰石Ultrogel、Q - Sepharose和Sephacryl S - 300柱层析相结合的方法，将α - L - 阿拉伯呋喃糖苷酶纯化至表观均一，最终比活性为6.46微摩尔/分钟/毫克。通过凝胶过滤法测得α - L - 阿拉伯吡喃糖苷酶的分子量为310 kDa，由四个相同的亚基组成（通过十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳[SDS - PAGE]测得每个亚基为77 kDa），通过凝胶过滤法和SDS - PAGE测得α - L - 阿拉伯呋喃糖苷酶的分子量为60 kDa。α - L - 阿拉伯吡喃糖苷酶和α - L - 阿拉伯呋喃糖苷酶的最佳活性分别在pH 5.5至6.0和40℃以及pH 4.5和45℃时表现。两种纯化的酶均受到Cu(2+)和对氯汞苯磺酸的强烈抑制。α - L - 阿拉伯吡喃糖苷酶对对硝基苯基 - α - L - 阿拉伯吡喃糖苷的作用最强，其次是人参皂苷Rb2。α - L - 阿拉伯呋喃糖苷酶对硝基苯基 - α - L - 阿拉伯呋喃糖苷的作用最强，其次是人参皂苷Rc。两种酶均不作用于对硝基苯基 - β - 半乳糖吡喃糖苷或对硝基苯基 - β - D - 岩藻糖吡喃糖苷。这些发现表明，这些纯化酶的生化性质和底物特异性与先前纯化的α - L - 阿拉伯糖苷酶不同。这是首次报道从厌氧双歧杆菌属中纯化出α - L - 阿拉伯吡喃糖苷酶。
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