Purification and characterization of α-L-arabinopyranosidase and α-L-arabinofuranosidase from Bifidobactetium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc
Purification and characterization of α-L-arabinopyranosidase and α-L-arabinofuranosidase from Bifidobactetium breve K-110, a human intestinal anaerobic bacterium metabolizing ginsenoside Rb2 and Rc
复制标题
DOI：
10.1128/aem.69.12.7116-7123.2003
复制
发表时间：
2003-12-01
影响因子：
4.4
通讯作者：
Kim, DH
中科院分区：
文献类型：
--
作者：
Shin, HY;Park, SY;Kim, DH
两种阿拉伯糖苷酶，α-L-阿拉伯吡喃糖苷酶（无EC编号）和α-L-阿拉伯呋喃糖苷酶（EC 3.2.1.55），从分离自人肠道微生物群落的糖苷代谢短双歧杆菌K-110中纯化。使用硫酸铵分级分离、DEAE-纤维素、丁基托伊皮翁、羟基磷灰石Ultrogel、QAE-纤维素和Sephacryl S-300 HR柱层析的组合，将α-L-阿拉伯吡喃糖苷酶纯化至表观均一性，最终比活性为8.81 μ mol/min/mg。使用硫酸铵分级分离、DEAE-纤维素、丁基托伊皮翁、羟基磷灰石Ultrogel、Q-Sepharose和Sephacryl S-300柱层析的组合将α-L-阿拉伯呋喃糖苷酶纯化至表观均一性，最终比活性为6.46 μ mol/min/mg。通过凝胶过滤发现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（每个亚基77 kDa，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳[SDS-PAGE]测量），并且通过凝胶过滤和SDS-PAGE发现α-L-阿拉伯吡喃糖苷酶的分子量为60 kDa。alpha-L-阿拉伯吡喃糖苷酶和alpha-L-阿拉伯呋喃糖苷酶的最适酶活分别为pH5.5 ~ 6.0和40 ℃，pH4.5和45 ℃。这两种纯化的酶都能被Cu ~（2+）和对氯汞苯磺酸（p-chlormercuryphenylsulfonic acid）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程度最大，其次是α-Rb 2。alpha-L-阿拉伯呋喃糖苷酶对对硝基苯基-alpha-L-阿拉伯呋喃糖苷的作用最大，其次是α-Rc。两种酶都不作用于对硝基苯基-β-吡喃半乳糖苷或对硝基苯基-β-D-吡喃岩藻糖苷。这些研究结果表明，这些纯化的酶的生化特性和底物特异性是不同于先前纯化的α-L-阿拉伯糖苷酶。这是第一次报道从厌氧双歧杆菌中纯化α-L-阿拉伯吡喃糖苷酶。
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 mumol/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 mumol/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-arabinopyranosidase 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 40degreesC and pH 4.5 and 45degreesC, respectively. Both purified enzymes were potently inhibited by Cu2+ 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.