To rationally engineer more efficient cellulolytic enzymes for cellulosic biomass deconstruction into sugars for biofuels production, it is necessary to better understand the complex enzyme-substrate interfacial interactions. Carbohydrate binding modules (CBM) are often associated with microbial surface-tethered cellulosomal or freely secreted cellulase enzymes to increase substrate accessibility. However, it is not well known how CBM recognize, bind, and dissociate from polysaccharide surfaces to facilitate efficient cellulolytic activity due to the lack of mechanistic understanding of CBM-substrate interactions. Our work outlines a general approach to methodically study the unbinding behavior of CBMs from model polysaccharide surfaces using single-molecule force spectroscopy. Here, we apply acoustic force spectroscopy (AFS) to probe a Clostridium thermocellum cellulosomal scaffoldin protein (CBM3a) and measure its dissociation from nanocellulose surfaces at physiologically relevant, low force loading rates. An automated microfluidic setup and methodology for uniform deposition of insoluble polysaccharides on the AFS chip surfaces is demonstrated. The rupture forces of wild-type CBM3a, and its Y67A mutant, unbinding from nanocellulose surface suggests distinct CBM binding conformations that can also explain the improved cellulolytic activity of cellulase tethered to CBM. Applying established dynamic force spectroscopy theory, the single-molecule unbinding rate at zero force is extrapolated and found to agree well with bulk equilibrium unbinding rates estimated independently using quartz crystal microbalance with dissipation monitoring. However, our results highlight the limitations of applying classical theory to explain the highly multivalent CBM-cellulose interactions seen at higher cellulose-CBM bond rupture forces (>15pN).

中文翻译：

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声力谱揭示碳水化合物结合模块的纤维素解结合行为的细微差异

为了合理设计更有效的纤维素分解酶，将纤维素生物质解构为用于生物燃料生产的糖，有必要更好地了解复杂的酶-底物界面相互作用。碳水化合物结合模块 (CBM) 通常与微生物表面束缚的纤维素酶或自由分泌的纤维素酶相关联，以增加底物可及性。然而，由于缺乏对 CBM-底物相互作用的机械理解，CBM 如何识别、结合和从多糖表面解离以促进有效的纤维素分解活性尚不清楚。我们的工作概述了使用单分子力谱从模型多糖表面有条不紊地研究 CBM 解绑行为的一般方法。在这里，我们应用声力谱 (AFS) 来探测热纤梭菌纤维素体支架蛋白 (CBM3a) 并在生理相关的低力加载速率下测量其与纳米纤维素表面的解离。展示了在 AFS 芯片表面上均匀沉积不溶性多糖的自动化微流体设置和方法。野生型 CBM3a 及其 Y67A 突变体的断裂力从纳米纤维素表面解离，表明不同的 CBM 结合构象也可以解释与 CBM 相连的纤维素酶的纤维素分解活性提高。应用已建立的动态力谱理论，外推零力下的单分子解结合率，发现与使用具有耗散监测的石英晶体微天平独立估计的体平衡解结合率非常吻合。然而，