NMR Analysis on Molecular Interaction of Lignin with Amino Acid Residues of Carbohydrate-Binding Module from Trichoderma reesei Cel7A

Abstract

Lignocellulosic biomass is anticipated to serve as a platform for green chemicals and fuels. Nonproductive binding of lignin to cellulolytic enzymes should be avoided for conversion of lignocellulose through enzymatic saccharification. Although carbohydrate-binding modules (CBMs) of cellulolytic enzymes strongly bind to lignin, the adsorption mechanism at molecular level is still unclear. Here, we report NMR-based analyses of binding sites on CBM1 of cellobiohydrolase I (Cel7A) from a hyper-cellulase-producing fungus, Trichoderma reesei, with cellohexaose and lignins from Japanese cedar (C-MWL) and Eucalyptus globulus (E-MWL). A method was established to obtain properly folded TrCBM1. Only TrCBM1 that was expressed in freshly transformed E. coli had intact conformation. Chemical shift perturbation analyses revealed that TrCBM1 adsorbed cellohexaose in highly specific manner via two subsites, flat plane surface and cleft, which were located on the opposite side of the protein surface. Importantly, MWLs were adsorbed at multiple binding sites, including the subsites, having higher affinity than cellohexaose. G6 and Q7 were involved in lignin binding on the flat plane surface of TrCBM1, while cellohexaose preferentially interacted with N29 and Q34. TrCBM1 used much larger surface area to bind with C-MWL than E-MWL, indicating the mechanisms of adsorption toward hardwood and softwood lignins are different.

NMR Elucidation of Non-productive Binding Sites of 13C-Labeled Lignin Models with Carbohydrate Binding Module of Cellobiohydrolase I for Efficient Biomass Conversion

“…NMR analysis of the 13 C-labeled lignin model compounds provided the rst direct evidence for the identi cation of binding atoms in the linear lignin chains. The results were in good agreement with our previous binding site analysis of the protein counterpart, TrCBM1 with MWL [17]. Herein, we provide direct evidence to reveal the interaction sites in the β-O-4 lignin substructure that bind to TrCBM1.…”

“…binding sites in proteins [16]. We previously performed CSP analysis to identify the binding amino acid residues in TrCBM1 with softwood and hardwood milled wood lignin (MWL) using 15 N-labeled TrCBM1 [17]. The results suggested that the aromatic rings in lignin participated in interactions with amino acid residues in TrCBM1, because the at plane surface including Y5, Y31, and Y32 in TrCBM1 was a main interaction site.…”

“…TrCBM1 was obtained after the addition of enterokinase and thrombin to cleave the His tag and GFP, respectively. We previously performed sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-ight mass spectrometry (MALDI-TOF-MS) and 2D 1 H- 15 N HSQC NMR to analyze the molecular mass and conformation of 15 Nlabeled TrCBM1 [17]. In this work, we performed SDS-PAGE and MALDI-TOF-MS to characterize unlabeled TrCBM1.…”

“…In recent work, we synthesized the unlabeled β-O-4 lignin oligomer model compound 4 ( Fig. 2) and fully characterized it by performing 1D 1 H NMR, 1D 13 C NMR, 2D 1 H-13 C HSQC, 2D 1 H-13 C HMBC, 2D 1 H-13 C long-range heteronuclear single quantum multiple bond correlation (LR-HSQMBC), 2D 1 H-1 H TOCSY, 2D 1 H-1 H EXSY and 2D 1 H-1 H ROESY [18]. We applied the protocol to synthesize three 13 C-labeled β-O-4 lignin oligomer model compounds.…”

Interaction of enzymes with lignocellulosic materials: causes, mechanism and influencing factors

“…Three types of interactions are involved in adsorption of enzyme onto lignin: (i) hydrophobic interactions (Ying et al 2018;Tokunaga et al 2019); (ii) electrostatic interactions (Nakagame et al 2011;Scott et al 2016), and (iii) hydrogen-bonding (Rahikainen et al 2013;Liu et al 2016). The CBDs of T. reesei cellulases, Cel7A and Cel5A were adsorbed onto lignin in significantly high amount (Yarbrough, et al 2015;Liu et al 2016) due to hydrophobic interaction.…”