In Vitro Reconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose
In Vitro Reconstitution of the Complete Clostridium thermocellum Cellulosome and Synergistic Activity on Crystalline Cellulose
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DOI：
10.1128/aem.07959-11
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发表时间：
2012-06-01
影响因子：
4.4
通讯作者：
Schwarz, Wolfgang H.
中科院分区：
文献类型：
--
作者：
Krauss, Jan;Zverlov, Vladimir V.;Schwarz, Wolfgang H.
对结晶纤维素有活性的人工纤维素酶复合物在体外从纤维素酶体酶和CipA支架蛋白的天然混合物重构。从C.热纤菌cipA突变体。它们通过锚定蛋白-黏连蛋白相互作用与纤维素体重新结合。使用具有或不具有碳水化合物结合模块（CBM）的具有一个至三个粘着蛋白和完整的CipA蛋白的重组产生的mini-CipA蛋白作为纤维素体骨架。粘着蛋白和锚定蛋白之间的结合自发发生。对可溶性和结晶纤维素化合物的水解活性表明，复合物的组成似乎并不依赖于CipA衍生的粘附素用于重建。结合似乎没有明显的局部偏好（与Coh 1和Coh 6的结合相等）。对结晶纤维素的协同作用随着支架中粘附素数量的增加而增加。在体外形成的复合物显示出12倍的协同作用的结晶基板（相比于未复合的组件）。用全尺寸CipA重构的纤维素酶体的活性达到天然纤维素酶体的80%。纳米颗粒表面的复合保留了蛋白质复合物的活性，提高了其稳定性。部分补充天然多纤维素酶体组分与三个选定的重组纤维素酶增强结晶纤维素的活性，并达到天然多纤维素酶体。这为体外复合物重构打开了可能性，这是朝着创建高效工程化纤维素酶的重要一步。
Artificial cellulase complexes active on crystalline cellulose were reconstituted in vitro from a native mix of cellulosomal enzymes and CipA scaffoldin. Enzymes containing dockerin modules for binding to the corresponding cohesin modules were prepared from culture supernatants of a C. thermocellum cipA mutant. They were reassociated to cellulosomes via dockerin-cohesin interaction. Recombinantly produced mini-CipA proteins with one to three cohesins either with or without the carbohydrate-binding module (CBM) and the complete CipA protein were used as the cellulosomal backbone. The binding between cohesins and dockerins occurred spontaneously. The hydrolytic activity against soluble and crystalline cellulosic compounds showed that the composition of the complex does not seem to be dependent on which CipA-derived cohesin was used for reconstitution. Binding did not seem to have an obvious local preference (equal binding to Coh1 and Coh6). The synergism on crystalline cellulose increased with an increasing number of cohesins in the scaffoldin. The in vitro-formed complex showed a 12-fold synergism on the crystalline substrate (compared to the uncomplexed components). The activity of reconstituted cellulosomes with full-size CipA reached 80% of that of native cellulosomes. Complexation on the surface of nanoparticles retained the activity of protein complexes and enhanced their stability. Partial supplementation of the native cellulosome components with three selected recombinant cellulases enhanced the activity on crystalline cellulose and reached that of the native cellulosome. This opens possibilities for in vitro complex reconstitution, which is an important step toward the creation of highly efficient engineered cellulases.