Highly efficient enzymatic saccharification of pretreated lignocellulose is a key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase that possesses carbohydrate-binding module 1 (TrCBM1) at the C-terminal domain. The nonproductive binding of TrCBM1 to lignin significantly decreases the enzymatic saccharification efficiency and increases the cost of biomass conversion because of the additionally required enzymes. Understanding the interaction mechanism between lignin and TrCBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated. Three types of 13C-labeled β-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H–13C heteronuclear single-quantum correlation (HSQC) spectra of the 13C-labeled lignin models confirmed that the three types of the 13C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The TrCBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13C-labeled β-O-4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to TrCBM1, whereas perturbations in the signals from aliphatic regions and methoxy groups were insignificant. These findings indicated that hydrophobic interactions and π–π stacking were dominating factors in nonproductive binding. The synthetic lignin models have two configurations whose terminal units were differently aligned and donated C(I) and C(II). The C(I) ring showed remarkable perturbation compared with the C(II), which indicated that the binding of TrCBM1 was markedly affected by the configuration of the lignin models. The long-chain lignin models (degree of polymerization (DP) 4.16–4.70) clearly bound to TrCBM1. The interactions of TrCBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. The CSP analysis using 13C-labeled β-O-4 lignin oligomer models enabled the identification of the TrCBM1 binding sites in lignins at the atomic level. This specific interaction analysis will provide insights for new molecular designs of cellulase having a controlled affinity to cellulose and lignin for a cost-effective biorefinery process.

中文翻译：

---

用纤维二糖水解酶 I 的碳水化合物结合模块对木质素模型的非生产性结合位点进行核磁共振分析

预处理的木质纤维素的高效酶糖化是实现木质纤维素生物精炼的关键步骤。里氏木霉分泌的纤维二糖水解酶 I (Cel7A) 是一种工业上使用的纤维素酶，在 C 末端结构域具有碳水化合物结合模块 1 (TrCBM1)。由于额外需要酶，TrCBM1 与木质素的非生产性结合显着降低了酶促糖化效率并增加了生物质转化的成本。了解木质素和 TrCBM1 之间的相互作用机制对于实现具有成本效益的生物燃料生产至关重要；然而，木质素中的结合位点尚未明确阐明。合成并表征了三种类型的 13C 标记的 β-O-4 木质素低聚物模型。13C 标记的木质素模型的 2D 1H–13C 异核单量子相关 (HSQC) 光谱证实，三种类型的 13C 标记正确地结合在 (1) 芳环和 β 位置，(2) α 位置， (3) 甲氧基。使用合成的 13C 标记的 β-O-4 木质素低聚物模型，通过观察 NMR 化学位移扰动 (CSP) 分析木质素中的 TrCBM1 结合位点。在与 TrCBM1 结合的低聚物的芳香族区域的信号中观察到明显的 CSP，而脂肪族区域和甲氧基的信号中的扰动是微不足道的。这些发现表明疏水相互作用和 π-π 堆积是非生产性结合的主要因素。合成木质素模型有两种配置，其末端单元排列不同，并捐赠了 C(I) 和 C(II)。与 C(II) 相比，C(I) 环显示出显着的扰动，这表明 TrCBM1 的结合受到木质素模型配置的显着影响。长链木质素模型（聚合度 (DP) 4.16-4.70）明显与 TrCBM1 结合。TrCBM1 与短链木质素模型（DP 2.64-3.12）的相互作用不显着，表明 DP 大于 4 是 TrCBM1 结合所必需的。使用 13C 标记的 β-O-4 木质素低聚物模型的 CSP 分析能够在原子水平上识别木质素中的 TrCBM1 结合位点。