Miller A W, Eklund S H, Robyt J F
Carbohydr Res. 1986 Mar 1;147(1):119-33. doi: 10.1016/0008-6215(86)85011-x.
A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.
相对于之前的方法，已优化了一系列步骤，包括葡聚糖酶处理、DEAE-纤维素色谱法、Sephadex G-200亲和色谱法以及DEAE-Trisacryl M色谱法，以获得碳水化合物含量低（1 - 100微克/毫克蛋白质）且比活性高（90 - 170 U/毫克蛋白质）的葡聚糖蔗糖酶制剂，产率为30 - 50%。在DEAE-纤维素色谱后没有果聚糖蔗糖酶，在Sephadex G-200色谱后未检测到葡聚糖酶。该方法可以扩大规模以生产克级量的纯化酶。纯化后的葡聚糖蔗糖酶最适pH为5.0 - 5.5，Km为12 - 16 mM，并且产生与纯化前相同的轻度分支的葡聚糖。纯化后的酶不会因添加葡聚糖而被激活，但在葡聚糖浓度约为0.1毫克/毫升的葡聚糖合成过程中，葡聚糖合成速率会突然增加。该酶有两种主要形式，分子量分别为177,000和158,000。177,000形式在培养上清液的新鲜制剂或纯化酶中占主导，而在任何一种制剂的储存过程中，158,000形式的量会以177,000形式为代价增加。
