Enzymatic hydrolysis of cellulose is a crucial step in lignocellulose sugar-platform biorefineries, and this process can be significantly inhibited by lignin-derived phenolics. This study investigated the inhibition of the enzymatic hydrolysis of cellulose by phenolics, focusing on its impact on Cel7A, a key enzyme involved in cellulose hydrolysis. Phenolics primarily inhibited the productive binding of Cel7A to cellulose, likely due to interference with catalytic domain (CD) binding. Surprisingly, phenolics increased Cel7A processivity. Kinetic modeling revealed that while phenolics increase Cel7A processivity, they decrease their association (k_on) and dissociation (k_off), and the dissociation step is considered as the limiting factor in hydrolysis in the presence of phenolics. Molecular dynamics simulations provided further insights, showing that phenolics form stable complexes with Cel7A through hydrogen bonds, hydrophobic interactions, and π-stacking. This binding altered Cel7A’s structure, resulting in a more enclosed catalytic tunnel and less flexible loops, likely accounting for the observed changes in processivity and k_off. Our study bridges the gap between interfacial kinetics and molecular simulations, thereby shedding light on the molecular mechanisms of phenolic inhibition. This knowledge paves the way for enzyme engineering strategies to enhance cellulase efficiency for biorefinery applications.

中文翻译：

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揭示木质素衍生酚类对纤维素酶水解的抑制作用：界面动力学和分子模拟


纤维素的酶水解是木质纤维素糖平台生物精炼厂的关键步骤，而木质素衍生的酚类物质可以显着抑制该过程。本研究研究了酚类物质对纤维素酶水解的抑制作用，重点关注其对 Cel7A（纤维素水解中涉及的关键酶）的影响。酚类物质主要抑制 Cel7A 与纤维素的有效结合，这可能是由于干扰催化结构域 (CD) 结合。令人惊讶的是，酚类物质提高了 Cel7A 的持续合成能力。动力学模型表明，虽然酚类物质提高了 Cel7A 的持续合成能力，但它们却降低了其缔合 (k _on ) 和解离 (k _off )，并且解离步骤被认为是水解的限制因素酚类物质的存在。分子动力学模拟提供了进一步的见解，表明酚类物质通过氢键、疏水相互作用和 π 堆积与 Cel7A 形成稳定的复合物。这种结合改变了 Cel7A 的结构，导致催化通道更加封闭，环路灵活性较差，这可能是观察到的持续合成能力和 k _off 变化的原因。我们的研究弥合了界面动力学和分子模拟之间的差距，从而揭示了酚类抑制的分子机制。这些知识为酶工程策略铺平了道路，以提高生物精炼应用中的纤维素酶效率。