Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.
Synergistic enhancement of cellulase pairs linked by consensus ankyrin repeats: Determination of the roles of spacing, orientation, and enzyme identity.
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DOI：
10.1002/prot.25047
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发表时间：
2016-08
期刊：
影响因子：
2.9
通讯作者：
Barrick D
中科院分区：
文献类型：
--
作者：
Cunha ES;Hatem CL;Barrick D
将生物质解构为小分子单糖是一种生产生物燃料的潜在方法，然而生物质的高度顽固性限制了这种方法的经济可行性。因此，为了实现生物燃料的大规模生产，对高效生物质降解的研究是必要的。通过提高纤维素酶之间的协同作用来增强纤维素分解活性，有望实现生物燃料的高产。在此，我们将来自极端微生物的纤维素酶对插入到超稳定的α - 螺旋共有锚蛋白重复结构域支架中。这种嵌合构建体使我们能够针对各种纤维素分解底物优化酶对的排列。我们发现内切纤维素酶结构域CelA（CA）和Cel12A（C12A）在锚蛋白重复序列的环境中，无论是三个还是四个重复间距，都具有协同作用。对于不同的底物，协同作用的程度有所不同。此外，C12A在CA的N端比相反的构建体具有更强的协同作用，尤其是针对滤纸。相比之下，我们发现这些酶与更大的外切纤维素酶CelK（CK）催化结构域串联时没有协同作用，这表明了酶的特性在协同增强中的重要性。此外，我们发现具有三个重复间距的内切纤维素酶CelD和CA对滤纸具有协同作用。重要的是，用相似轮廓长度的无序连接子连接CA和C12A时，没有协同增强作用，这表明协同作用是由这些结构域与折叠的锚蛋白重复序列连接产生的。这些结果表明，锚蛋白阵列可用于改变酶之间的间距和方向，有助于设计和优化人工纤维素体，为酶促纤维素降解的协同增强提供一种新的结构。
Biomass deconstruction to small simple sugars is a potential approach to biofuels production, however the highly recalcitrant nature of biomass limits the economic viability of this approach. Thus, research on efficient biomass degradation is necessary to achieve large-scale production of biofuels. Enhancement of cellulolytic activity by increasing synergism between cellulase enzymes holds promise in achieving high-yield biofuels production. Here we have inserted cellulase pairs from extremophiles into hyper-stable α-helical consensus ankyrin repeat domain scaffolds. Such chimeric constructs allowed us to optimize arrays of enzyme pairs against a variety of cellulolytic substrates. We found that endocellulolytic domains CelA (CA) and Cel12A (C12A) act synergistically in the context of ankyrin repeats, with both three and four repeat spacing. The extent of synergy differs for different substrates. Also, having C12A N-terminal to CA provides greater synergy than the reverse construct, especially against filter paper. In contrast, we do not see synergy for these enzymes in tandem with CelK (CK) catalytic domain, a larger exocellulase, demonstrating the importance of enzyme identity in synergistic enhancement. Furthermore, we found endocellulases CelD and CA with three repeat spacing to act synergistically against filter paper. Importantly, connecting CA and C12A with a disordered linker of similar contour length, shows no synergistic enhancement, indicating that synergism results from connecting these domains with folded ankyrin repeats. These results show that ankyrin arrays can be used to vary spacing and orientation between enzymes, helping to design and optimize artificial cellulosomes, providing a novel architecture for synergistic enhancement of enzymatic cellulose degradation.
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DOI：
10.1196/annals.1419.002
发表时间：
2008-01-01
期刊：
INCREDIBLE ANAEROBES: FROM PHYSIOLOGY TO GENOMICS TO FUELS
影响因子：
--
作者：
Doi, Roy H.
通讯作者：
Doi, Roy H.
影响因子：
5.7
作者：
Alzari, PM;Souchon, H;Dominguez, R
通讯作者：
Dominguez, R
影响因子：
17.3
作者：
BAYER, EA;MORAG, E;LAMED, R
通讯作者：
LAMED, R
影响因子：
4.4
作者：
Cunha, Eva S.;Hatem, Christine L.;Barrick, Doug
通讯作者：
Barrick, Doug
影响因子：
5
作者：
Cheng, Ya-Shan;Ko, Tzu-Ping;Guo, Rey-Ting
通讯作者：
Guo, Rey-Ting