Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
Cellobiohydrolase 1 from Trichoderma reesei degrades cellulose in single cellobiose steps.
复制标题
来自Trichoderma Reesei的Bobiobiohydrollose 1在单核生物步骤中降解了纤维素。
DOI：
10.1038/ncomms10149
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发表时间：
2015-12-10
影响因子：
16.6
通讯作者：
Lang MJ
中科院分区：
文献类型：
--
作者：
Brady SK;Sreelatha S;Feng Y;Chundawat SP;Lang MJ
里氏木霉的纤维素生物水解酶1 （TrCel7A）将纤维素水解成纤维素二糖。虽然酶技术已被确立为生物燃料生产中有前途的工具，但对马达的机械作用的清晰理解尚未揭示。在这里，我们开发了一种基于光学镊子的单分子（SM）运动测定方法，用于精确跟踪TrCel7A。直接观察降解过程中的运动，可以发现在1纳米尺度上的递进运行和不同的步骤。我们的研究表明，TrCel7A不受机械限制，可以在20pn负载下工作，并且在辅助下可以加速。依赖于温度的动力学研究确定了基本步进循环的能量需求，其中可能包括来自糖苷键和其他来源的能量。通过对分离的TrCel7A结构域的SM测量，我们确定仅催化结构域就足以进行过程运动，从而深入了解TrCel7A的分子运动机制。纤维素生物水解酶是一种很有前途的生物燃料生产工具，它能将纤维素水解成纤维素二糖。在这里，作者使用光学镊子展示了来自里氏木霉的纤维生物水解酶1在中等负荷下的渐进功能，并且可能使用多种能量来推动纤维素纤维的每一步。
Cellobiohydrolase 1 from Trichoderma reesei (TrCel7A) processively hydrolyses cellulose into cellobiose. Although enzymatic techniques have been established as promising tools in biofuel production, a clear understanding of the motor's mechanistic action has yet to be revealed. Here, we develop an optical tweezers-based single-molecule (SM) motility assay for precision tracking of TrCel7A. Direct observation of motility during degradation reveals processive runs and distinct steps on the scale of 1 nm. Our studies suggest TrCel7A is not mechanically limited, can work against 20 pN loads and speeds up when assisted. Temperature-dependent kinetic studies establish the energy requirements for the fundamental stepping cycle, which likely includes energy from glycosidic bonds and other sources. Through SM measurements of isolated TrCel7A domains, we determine that the catalytic domain alone is sufficient for processive motion, providing insight into TrCel7A's molecular motility mechanism. Cellobiohydrolases are promising tools in biofuel production by hydrolysing cellulose into cellobiose. Here the authors use optical tweezers to show that Cellobiohydrolase 1 from Tricodermia reesei functions processively against moderate load, and likely uses multiple energy sources to fuel each step along the cellulose fibre.