NMR elucidation of nonproductive binding sites of lignin models with carbohydrate-binding module of cellobiohydrolase I

Abstract

Abstract Background 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 ( Tr CBM1) at the C-terminal domain. The nonproductive binding of Tr CBM1 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 Tr CBM1 is essential for realizing a cost-effective biofuel production; however, the binding sites in lignin have not been clearly elucidated. Results Three types of 13 C-labeled β- O -4 lignin oligomer models were synthesized and characterized. The 2D 1 H– 13 C heteronuclear single-quantum correlation (HSQC) spectra of the 13 C-labeled lignin models confirmed that the three types of the 13 C labels were correctly incorporated in the (1) aromatic rings and β positions, (2) α positions, and (3) methoxy groups, respectively. The Tr CBM1-binding sites in lignin were analyzed by observing NMR chemical shift perturbations (CSPs) using the synthetic 13 C-labeled β- O -4 lignin oligomer models. Obvious CSPs were observed in signals from the aromatic regions in oligomers bound to Tr CBM1, 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 Tr CBM1 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 Tr CBM1. The interactions of Tr CBM1 with the short-chain lignin models (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for Tr CBM1 binding. Conclusion The CSP analysis using 13 C-labeled β- O -4 lignin oligomer models enabled the identification of the Tr CBM1 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.

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