Asem K G, Kenney A C, Cole J A
Arch Biochem Biophys. 1986 Feb 1;244(2):607-18. doi: 10.1016/0003-9861(86)90629-6.
Two methods were used to purify the bifunctional extracellular enzyme sucrose: (1-6)- and (1-3)-alpha-D-glucan-6-alpha-D-glucosyltransferase (EC 2.4.1.5; dextransucrase) from continuous cultures of a serotype c strain of Streptococcus mutans. The first method, based on a previously published report, involved Sepharose 6B gel filtration and DEAE cellulose anion exchange chromatography. This resulted in a dextransucrase preparation with an apparent molecular mass of 162 kDa and a specific activity of 125 mg of glucan formed from sucrose h-1 (mg of protein)-1, at 37 degrees C. It was almost homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The ratio of carbohydrate to protein was 0.14 and the recovery was 14% relative to the total glucosyltransferase activity in the original culture fluid. In the subsequently preferred method, hydroxyapatite-Ultrogel was used to purify dextransucrase with a 24% yield. The specific activity, 197 mg of glucan formed h-1 (mg of protein)-1, was the highest yet reported and this preparation contained less than 0.5 glucose-equivalent per subunit of molecular mass 162 kDa. Dextransucrase is therefore not a glycoprotein. Exogenous dextran stimulated activity, but was not essential for activity. The purified protein slowly degraded to multiple lower molecular mass forms during storage at 4 degrees C and 87% of the activity was lost after 20 days. The molecular mass of the most prominent, active degradation product was 140 kDa, similar to that of one of the multiple forms of dextransucrase detected in other laboratories. Preparations in which either the 140-kDa or the 162-kDa species predominated catalyzed the synthesis of a water-soluble glucan with sucrose alone, but catalyzed that of an insoluble glucan with sucrose and a high concentration of either (NH4)2SO4 or polyethylene glycol. The water-insoluble glucan was shown to lack sequences of 1,3-alpha-linked glycosyl residues typical of the insoluble glucan, mutan, which has been implicated in dental caries. We conclude that mutan is synthesized by the concerted action of two independent glucosyltransferases rather than by interconvertible forms of a single enzyme, as was proposed previously.
采用两种方法从变形链球菌血清型c菌株的连续培养物中纯化双功能胞外酶蔗糖：(1-6)-和(1-3)-α-D-葡聚糖-6-α-D-葡萄糖基转移酶（EC 2.4.1.5；葡聚糖蔗糖酶）。第一种方法基于先前发表的报告，包括琼脂糖6B凝胶过滤和DEAE纤维素阴离子交换色谱。这得到了一种葡聚糖蔗糖酶制剂，其表观分子量为162 kDa，在37℃下由蔗糖形成葡聚糖的比活性为125 mg·h⁻¹·(mg蛋白)⁻¹。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳判断，它几乎是纯的。碳水化合物与蛋白质的比率为0.14，相对于原始培养液中总葡糖基转移酶活性，回收率为14%。在随后更优的方法中，使用羟基磷灰石-优特凝胶纯化葡聚糖蔗糖酶，产率为24%。比活性为197 mg·h⁻¹·(mg蛋白)⁻¹，是迄今报道的最高值，并且该制剂每162 kDa分子质量亚基含有的葡萄糖当量少于0.5。因此，葡聚糖蔗糖酶不是糖蛋白。外源葡聚糖刺激活性，但对活性不是必需的。纯化后的蛋白质在4℃储存期间缓慢降解为多种较低分子量形式，20天后87%的活性丧失。最突出的活性降解产物的分子量为140 kDa，与其他实验室检测到的葡聚糖蔗糖酶多种形式之一相似。以140 kDa或162 kDa为主的制剂单独用蔗糖催化合成水溶性葡聚糖，但用蔗糖和高浓度的硫酸铵或聚乙二醇催化合成不溶性葡聚糖。结果表明，这种水不溶性葡聚糖缺乏与龋齿有关的不溶性葡聚糖变聚糖典型的1,3-α-连接糖基残基序列。我们得出结论，变聚糖是由两种独立的葡糖基转移酶协同作用合成的，而不是如先前提出的由单一酶的可相互转化形式合成。
