Action of designer cellulosomes on homogeneous versus complex substrates -: Controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin
Action of designer cellulosomes on homogeneous versus complex substrates -: Controlled incorporation of three distinct enzymes into a defined trifunctional scaffoldin
复制标题
DOI：
10.1074/jbc.m414449200
复制
发表时间：
2005-04-22
影响因子：
4.8
通讯作者：
Bayer, EA
中科院分区：
文献类型：
--
作者：
Fierobe, HP;Mingardon, F;Bayer, EA
在最近的工作中(Fierobe，H.P.，Bayer，E.A.，Tardif，C.，Chujzek，M.，Mechaly，A.，Belai“ch，A.，Lamed，R.，Shoham，Y.和Belaich，J.-P.(2002)J.Biol.化学。49621,49630)，我们报道了一套完整的已定义的“双功能”嵌合纤维素体的自组装。每个复合体包含以下内容：(I)具有一个纤维素结合模块和两个具有不同特异性的粘附素的嵌合支架蛋白和(Ii)两个纤维素酶，每个纤维素酶带有一个与其中一个不同粘附素互补的Dockerin。这种方法允许受控地将所需的酶整合到预定化学计量比和拓扑结构的多蛋白质复合体中。双功能设计者纤维素体在顽固底物上观察到的增强协同作用归因于两个主要因素：底物靶向和两个催化组分的接近。在本工作中，通过开发第三种发散型粘附素-对接蛋白装置，放大了先前描述的嵌合纤维素体的能力。得到的三功能设计者纤维素体在均相和复合底物(分别为微晶纤维素和稻草)上进行了测试，发现比相应的游离酶或双功能体系的活性要高得多。结果表明，在纤维素体降解纤维素的过程中，两种重要的纤维素体酶(-48和-9糖苷水解酶)之间的协同作用起着至关重要的作用，每个复合体中一个显性的48家族内切葡聚糖酶足以达到最佳的协同活性水平。此外，纤维素体嵌合体内纤维素酶和来自不同微生物的半纤维素酶之间的合作也实现了，导致了在复合底物上具有增强活性的三功能复合体。
In recent work (Fierobe, H.- P., Bayer, E. A., Tardif, C., Czjzek, M., Mechaly, A., Belai " ch, A., Lamed, R., Shoham, Y., and Belaich, J.- P. ( 2002) J. Biol. Chem. 277, 49621 49630), we reported the self-assembly of a comprehensive set of defined " bifunctional" chimeric cellulosomes. Each complex contained the following: ( i) a chimeric scaffoldin possessing a cellulose-binding module and two cohesins of divergent specificity and (ii) two cellulases, each bearing a dockerin complementary to one of the divergent cohesins. This approach allowed the controlled integration of desired enzymes into a multiprotein complex of predetermined stoichiometry and topology. The observed enhanced synergy on recalcitrant substrates by the bifunctional designer cellulosomes was ascribed to two major factors: substrate targeting and proximity of the two catalytic components. In the present work, the capacity of the previously described chimeric cellulosomes was amplified by developing a third divergent cohesin-dockerin device. The resultant trifunctional designer cellulosomes were assayed on homogeneous and complex substrates ( microcrystalline cellulose and straw, respectively) and found to be considerably more active than the corresponding free enzyme or bifunctional systems. The results indicate that the synergy between two prominent cellulosomal enzymes ( from the family-48 and -9 glycoside hydrolases) plays a crucial role during the degradation of cellulose by cellulosomes and that one dominant family-48 processive endoglucanase per complex is sufficient to achieve optimal levels of synergistic activity. Furthermore cooperation within a cellulosome chimera between cellulases and a hemicellulase from different microorganisms was achieved, leading to a trifunctional complex with enhanced activity on a complex substrate.