Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode
Effect of Linker Length and Dockerin Position on Conversion of a Thermobifida fusca Endoglucanase to the Cellulosomal Mode
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DOI：
10.1128/aem.01241-09
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发表时间：
2009-12-01
影响因子：
4.4
通讯作者：
Bayer, Edward A.
中科院分区：
文献类型：
--
作者：
Caspi, Jonathan;Barak, Yoav;Bayer, Edward A.
我们一直在开发青海高温双歧杆菌的纤维素酶作为模型，以探索从游离纤维素酶系统到纤维素体模式的转化。在以前的工作中，6种青霉纤维素酶中的3种(内切葡聚糖酶Cel6A和外切葡聚糖酶Cel6B和Cel48A)通过用Dockerin取代它们的纤维素结合模块(CBM)进行了转化，得到的重组纤维素化的酶与CBM一起被整合到含有粘附素的嵌合支架蛋白(S)中。将得到的设计者纤维素体的活性与同等的野生型酶混合物进行了比较。在本工作中，第四个青霉纤维素酶Cel5A配备了一个Dockerin和不同长度的中间接头片段，以评估它们对简单的单酶和双酶设计的纤维素体复合体整体活性的贡献。结果表明，纤维素结合在结晶纤维素底物的降解中起主要作用。转化后的Cel5A内切葡聚糖酶和转化后的Cel48A外切葡聚糖酶的结合对最顽固的纤维素底物(Avicel)也显示出可测量的邻近效应。催化模块和Dockerin之间的连接子的长度对活性几乎没有影响。然而，将Dockerin定位在酶的相反(C-末端)一侧，与Dockerin在大多数纤维素体酶上的通常位置一致，导致了增强的协同反应。这些结果促进了更复杂的多酶设计纤维素体的发展，最终可能应用于改善植物细胞壁生物量的降解。
We have been developing the cellulases of Thermobifida fusca as a model to explore the conversion from a free cellulase system to the cellulosomal mode. Three of the six T. fusca cellulases (endoglucanase Cel6A and exoglucanases Cel6B and Cel48A) have been converted in previous work by replacing their cellulose-binding modules (CBMs) with a dockerin, and the resultant recombinant "cellulosomized" enzymes were incorporated into chimeric scaffolding proteins that contained cohesin(s) together with a CBM. The activities of the resultant designer cellulosomes were compared with an equivalent mixture of wild-type enzymes. In the present work, a fourth T. fusca cellulase, Cel5A, was equipped with a dockerin and intervening linker segments of different lengths to assess their contribution to the overall activity of simple one-and two-enzyme designer cellulosome complexes. The results demonstrated that cellulose binding played a major role in the degradation of crystalline cellulosic substrates. The combination of the converted Cel5A endoglucanase with the converted Cel48A exoglucanase also exhibited a measurable proximity effect for the most recalcitrant cellulosic substrate (Avicel). The length of the linker between the catalytic module and the dockerin had little, if any, effect on the activity. However, positioning of the dockerin on the opposite (C-terminal) side of the enzyme, consistent with the usual position of dockerins on most cellulosomal enzymes, resulted in an enhanced synergistic response. These results promote the development of more complex multienzyme designer cellulosomes, which may eventually be applied for improved degradation of plant cell wall biomass.