Purification and characterization of cell-associated glucosyltransferase synthesizing water-insoluble glucan from serotype c Streptococcus mutans.
Purification and characterization of cell-associated glucosyltransferase synthesizing water-insoluble glucan from serotype c Streptococcus mutans.
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从 c 血清型变形链球菌中合成水不溶性葡聚糖的细胞相关葡糖基转移酶的纯化和表征。
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发表时间：
1989
期刊：
影响因子：
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通讯作者：
T. Koga
中科院分区：
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作者：
S. Hamada;T. Horikoshi;T. Minami;N. Okahashi;T. Koga
变形链球菌菌株（血清型c，e和f）被发现具有高水平的葡萄糖基转移酶（GT酶）的活性，无论是细胞相关的，并在培养基中，生长在无蔗糖或含蔗糖的肉汤培养基。S.通过在25 ℃下用8 M-尿素处理1小时有效地提取了变形杆菌MT 8148（血清型c）。约95%的GTase活性通过该处理被溶解。粗提物经DEAE-Sephacel和羟基磷灰石柱层析纯化。为了比较，还通过硫酸铵沉淀、层析聚焦和羟基磷灰石层析从同一菌株的培养上清中纯化了细胞外GTase。通过SDS-PAGE测定，细胞相关和细胞外GT酶蛋白的分子量相似（156 kDa）。然而，最大GT酶活性的最适pH是不同的：pH 6.7至7.0的细胞相关酶和pH 5.5至6.5的胞外酶。来自蔗糖的细胞相关GT酶的产物几乎完全是水不溶性葡聚糖。另一方面，胞外GTase主要从蔗糖中产生水溶性葡聚糖。这表明合成水不溶性葡聚糖的GT酶主要以细胞结合形式存在于血清型c S中。变异人不溶性葡聚糖合成的细胞相关的GT酶从蔗糖没有增加引物葡聚糖T10的反应混合物。细胞外和细胞相关的GT酶是免疫无关的，通过ELISA使用单克隆抗体。
Strains of Streptococcus mutans (serotypes c, e and f) were found to possess high levels of glucosyltransferase (GTase) activity, both cell-associated and in the culture medium, when grown in either sucrose-free or sucrose-containing broth media. The cell-associated GTase of S. mutans MT8148 (serotype c) was effectively extracted by treatment with 8 M-urea at 25 degrees C for 1 h. Approximately 95% of the GTase activity was solubilized by this treatment. The crude extract was purified by DEAE-Sephacel and hydroxylapatite column chromatography. For comparison, extracellular GTase was also purified from the culture supernatant of the same strain by ammonium sulphate precipitation, chromatofocusing and hydroxylapatite chromatography. The molecular masses of the cell-associated and extracellular GTase proteins were similar (156 kDa) as determined by SDS-PAGE. However, the pH optima for maximum GTase activity were different: pH 6.7 to 7.0 for the cell-associated enzyme and pH 5.5 to 6.5 for the extracellular enzyme. The product of cell-associated GTase from sucrose was almost exclusively water-insoluble glucan. On the other hand, extracellular GTase produced mainly water-soluble glucan from sucrose. This indicates that GTase synthesizing water-insoluble glucan is present primarily in a cell-associated form in serotype c S. mutans. Insoluble glucan synthesis by the cell-associated GTase from sucrose was not enhanced by addition of primer dextran T10 to the reaction mixture. The extracellular and cell-associated GTases were immunologically unrelated as determined by ELISA using monoclonal antibodies.