Chia J S, Hsieh C C, Yang C S, Chen J Y
Department of Bacteriology, College of Medicine, National Taiwan University, Taipei, Republic of China.
Zhonghua Min Guo Wei Sheng Wu Ji Mian Yi Xue Za Zhi. 1995 Feb;28(1):1-12.
Streptococcus mutants constitutively expresses three glucosyltransferases (GTFs), i.e., GtfB, GtfC, and GtfD, which synthesize glucan polymers from sucrose. Two genetically constructed mutants of S. mutans which stably expressed either the cell-associated or the extracellular GTFs were selected for purification and characterization of these enzymes. The cell-associated GtfB and GtfC from strain GS-5DD lacking the gtfD gene expression were extracted by urea, renatured by dialysis in sodium phosphate buffer and then separated from the other wall-associated components by column chromatography. The extracellular GtfD was purified from the culture supernatant of strain NHS1 lacking gtfB and gtfC gene expression. The molecular weights of the purified GTFs was similar (150-160 kDa), as determined by SDS-polyacrylamide gel electrophoresis. The GtfB/C preparation synthesized primarily water-insoluble glucan in a primer independent manner. However, the presence of the dextran enhanced the enzymatic activities of the GtfB/C. GtfD synthesized water-soluble glucan exclusively in a primer dependent manner. Purified GtfD had a pH optimum of 5.5, and a K(m) value of 4.35 mM for sucrose. These results indicated that the mutated strains served as an efficient and specific host to obtain native GTFs.
变形链球菌组成性表达三种葡糖基转移酶（GTFs），即GtfB、GtfC和GtfD，它们可从蔗糖合成葡聚糖聚合物。选择了两种稳定表达细胞相关或细胞外GTFs的变形链球菌基因构建突变体，用于这些酶的纯化和特性鉴定。通过尿素提取缺乏gtfD基因表达的GS-5DD菌株的细胞相关GtfB和GtfC，在磷酸钠缓冲液中透析复性，然后通过柱色谱与其他细胞壁相关成分分离。从缺乏gtfB和gtfC基因表达的NHS1菌株的培养上清液中纯化细胞外GtfD。通过SDS-聚丙烯酰胺凝胶电泳测定，纯化的GTFs的分子量相似（150-160 kDa）。GtfB/C制剂主要以不依赖引物的方式合成水不溶性葡聚糖。然而，葡聚糖的存在增强了GtfB/C的酶活性。GtfD仅以依赖引物的方式合成水溶性葡聚糖。纯化的GtfD最适pH为5.5，对蔗糖的K(m)值为4.35 mM。这些结果表明，突变菌株是获得天然GTFs的有效且特异的宿主。
