Lee LanNa, Saxena Deepak, Stotzky G
Laboratory of Microbial Ecology, Department of Biology, New York University, New York, NY 10003, USA.
Appl Environ Microbiol. 2003 Jul;69(7):4111-5. doi: 10.1128/AEM.69.7.4111-4115.2003.
Bacillus thuringiensis subsp. israelensis produces parasporal insecticidal crystal proteins (ICPs) that have larvicidal activity against some members of the order Diptera, such as blackflies and mosquitoes. Hydrolysis of the ICPs in the larval gut results in four major proteins with a molecular mass of 27, 65, 128, and 135 kDa. Toxicity is caused by synergistic interaction between the 25-kDa protein (proteolytic product of the 27-kDa protein) and one or more of the higher-molecular-mass proteins. Equilibrium adsorption of the proteins on the clay minerals montmorillonite and kaolinite, which are homoionic to various cations, was rapid (<30 min for maximal adsorption), increased with protein concentration and then reached a plateau (68 to 96% of the proteins was adsorbed), was significantly lower on kaolinite than on montmorillonite, and was not significantly affected by the valence of the cation to which the clays were homoionic. Binding of the toxins decreased as the pH was increased from 6 to 11, and there was 35 to 66% more binding in phosphate buffer at pH 6 than in distilled water at pH 6 or 7.2. Only 2 to 12% of the adsorbed proteins was desorbed by two washes with water; additional washings desorbed no more toxins, indicating that they were tightly bound. Formation of clay-toxin complexes did not alter the structure of the proteins, as indicated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the equilibrium supernatants and desorption washes and by dot blot enzyme-linked immunosorbent assay of the complexes, which was confirmed by enhanced chemiluminescence Western blot analysis. Free and clay-bound toxins resulted in 85 to 100% mortality of the mosquito Culex pipiens. Persistence of the bound toxins in nonsterile water after 45 days was significantly greater (mortality of 63% +/- 12.7%) than that of the free toxins (mortality of 25% +/- 12.5%).
苏云金芽孢杆菌以色列亚种产生伴孢杀虫晶体蛋白（ICPs），这些蛋白对双翅目昆虫的某些种类具有杀幼虫活性，如蚋和蚊子。幼虫肠道中的ICPs水解后产生四种主要蛋白质，分子量分别为27、65、128和135 kDa。毒性是由25 kDa蛋白质（27 kDa蛋白质的蛋白水解产物）与一种或多种高分子量蛋白质之间的协同相互作用引起的。这些蛋白质在与各种阳离子同离子的粘土矿物蒙脱石和高岭石上的平衡吸附很快（最大吸附在30分钟内），随蛋白质浓度增加，然后达到平台期（68%至96%的蛋白质被吸附），在高岭石上的吸附明显低于蒙脱石，且不受粘土同离子阳离子价态的显著影响。毒素的结合随着pH从6增加到11而降低，在pH 6的磷酸盐缓冲液中的结合比在pH 6或7.2的蒸馏水中多35%至66%。用水洗涤两次后，只有2%至12%的吸附蛋白质被解吸；额外洗涤不再解吸出更多毒素，表明它们结合紧密。如平衡上清液和解吸洗涤液的十二烷基硫酸钠-聚丙烯酰胺凝胶电泳以及复合物的斑点印迹酶联免疫吸附测定所示，粘土-毒素复合物的形成并未改变蛋白质的结构，增强化学发光免疫印迹分析证实了这一点。游离和粘土结合的毒素导致致倦库蚊85%至100%的死亡率。45天后，结合毒素在非无菌水中的持久性明显高于游离毒素（死亡率为63%±12.7%，而游离毒素死亡率为25%±12.5%）。
