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

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 (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. Results 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. Conclusion 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.

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NMR Elucidation of Nonproductive Binding Sites of Lignin Models with Carbohydrate-Binding Module of Cellobiohydrolase I

10.21203/rs.3.rs-36005/v2 ◽

2020 ◽

Author(s):

Yuki Tokunaga ◽

Takashi Nagata ◽

Keiko Kondo ◽

Masato Katahira ◽

Takashi Watanabe

Keyword(s):

Binding Sites ◽

Specific Interaction ◽

Enzymatic Saccharification ◽

Cost Effective ◽

Biofuel Production ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Cellobiohydrolase I ◽

Methoxy Groups ◽

Nonproductive Binding

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 (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.Results: 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.Conclusion: 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.

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

10.21203/rs.3.rs-36005/v1 ◽

2020 ◽

Author(s):

Yuki Tokunaga ◽

Takashi Nagata ◽

Keiko Kondo ◽

Masato Katahira ◽

Takashi Watanabe

Keyword(s):

Binding Sites ◽

Hydrophobic Interactions ◽

Biomass Conversion ◽

Specific Interaction ◽

Enzymatic Saccharification ◽

Cost Effective ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Cellobiohydrolase I ◽

Methoxy Groups

Abstract Background: Highly efficient enzymatic saccharification of pretreated lignocellulose is a primary key step in achieving lignocellulosic biorefinery. Cellobiohydrolase I (Cel7A) secreted by Trichoderma reesei is an industrially used cellulase possessing carbohydrate binding module 1 (TrCBM1) as the C-terminal domain. Non-productive binding of TrCBM1 to lignin significantly decreases enzymatic saccharification efficiency and enhance cost of biomass conversion due to required additional enzymes. Understanding of the interaction mechanism between lignin and TrCBM1 is essentially required to realize cost-effective biofuels production, but the binding sites in lignin have not been clearly elucidated. Results: Three types of 13C-labeled b-O-4 lignin oligomer models were synthesized and characterized. The 2D 1H-13C HSQC spectra of the 13C-labeled lignin models exhibited that 13C-labels were correctly incorporated in the (1) aromatic rings and b positions, (2) a 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 b-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. This indicated that hydrophobic interactions and p–p stacking were dominating factors in non-productive 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 C(II), which indicated that binding of TrCBM1 is evidently affected by configuration of lignin models. Long-chain lignins (DP 4.16–4.70) clearly bound to TrCBM1. Interactions with short-chain lignins (DP 2.64–3.12) were insignificant, indicating that a DP greater than 4 was necessary for TrCBM1 binding. Conclusion: The CSP analysis using 13C-labeled b-O-4 lignin oligomer models enabled us to identify TrCBM1 binding sites in lignin at the atomic level. This specific interaction analysis will lead to new molecular design of cellulase having controlled affinity to cellulose and lignin for cost-effective biorefinery process.

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Domain Analysis of a Modular α-l-Arabinofuranosidase with a Unique Carbohydrate Binding Strategy from the Fiber-Degrading Bacterium Fibrobacter succinogenes S85

Journal of Bacteriology ◽

10.1128/jb.00503-10 ◽

2010 ◽

Vol 192 (20) ◽

pp. 5424-5436 ◽

Cited By ~ 13

Author(s):

Shosuke Yoshida ◽

Charles W. Hespen ◽

Robert L. Beverly ◽

Roderick I. Mackie ◽

Isaac K. O. Cann

Keyword(s):

Plant Cell Wall ◽

Catalytic Efficiency ◽

Glycoside Hydrolases ◽

Site Directed Mutagenesis ◽

Biofuel Production ◽

Carbohydrate Binding ◽

Fibrobacter Succinogenes ◽

Carbohydrate Binding Module ◽

Degrading Bacterium ◽

Catalytic Module

ABSTRACT Family 43 glycoside hydrolases (GH43s) are known to exhibit various activities involved in hemicellulose hydrolysis. Thus, these enzymes contribute to efficient plant cell wall degradation, a topic of much interest for biofuel production. In this study, we characterized a unique GH43 protein from Fibrobacter succinogenes S85. The recombinant protein showed α-l-arabinofuranosidase activity, specifically with arabinoxylan. The enzyme is, therefore, an arabinoxylan arabinofuranohydrolase (AXH). The F. succinogenes AXH (FSUAXH1) is a modular protein that is composed of a signal peptide, a GH43 catalytic module, a unique β-sandwich module (XX domain), a family 6 carbohydrate-binding module (CBM6), and F. succinogenes-specific paralogous module 1 (FPm-1). Truncational analysis and site-directed mutagenesis of the protein revealed that the GH43 domain/XX domain constitute a new form of carbohydrate-binding module and that residue Y484 in the XX domain is essential for binding to arabinoxylan, although protein structural analyses may be required to confirm some of the observations. Kinetic studies demonstrated that the Y484A mutation leads to a higher k cat for a truncated derivative of FSUAXH1 composed of only the GH43 catalytic module and the XX domain. However, an increase in the Km for arabinoxylan led to a 3-fold decrease in catalytic efficiency. Based on the knowledge that most XX domains are found only in GH43 proteins, the evolutionary relationships within the GH43 family were investigated. These analyses showed that in GH43 members with a XX domain, the two modules have coevolved and that the length of a loop within the XX domain may serve as an important determinant of substrate specificity.

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The family 21 carbohydrate-binding module of glucoamylase from Rhizopus oryzae consists of two sites playing distinct roles in ligand binding

Biochemical Journal ◽

10.1042/bj20051982 ◽

2006 ◽

Vol 396 (3) ◽

pp. 469-477 ◽

Cited By ~ 27

Author(s):

Wei-I Chou ◽

Tun-Wen Pai ◽

Shi-Hwei Liu ◽

Bor-Kai Hsiung ◽

Margaret D.-T. Chang

Keyword(s):

Ligand Binding ◽

Binding Sites ◽

Catalytic Domain ◽

Rhizopus Oryzae ◽

Structural Information ◽

Molecular Model ◽

Site Directed Mutagenesis ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Ligand Binding Sites

The starch-hydrolysing enzyme GA (glucoamylase) from Rhizopus oryzae is a commonly used glycoside hydrolase in industry. It consists of a C-terminal catalytic domain and an N-terminal starch-binding domain, which belong to the CBM21 (carbohydrate-binding module, family 21). In the present study, a molecular model of CBM21 from R. oryzae GA (RoGACBM21) was constructed according to PSSC (progressive secondary structure correlation), modified structure-based sequence alignment, and site-directed mutagenesis was used to identify and characterize potential ligand-binding sites. Our model suggests that RoGACBM21 contains two ligand-binding sites, with Tyr32 and Tyr67 grouped into site I, and Trp47, Tyr83 and Tyr93 grouped into site II. The involvement of these aromatic residues has been validated using chemical modification, UV difference spectroscopy studies, and both qualitative and quantitative binding assays on a series of RoGACBM21 mutants. Our results further reveal that binding sites I and II play distinct roles in ligand binding, the former not only is involved in binding insoluble starch, but also facilitates the binding of RoGACBM21 to long-chain soluble polysaccharides, whereas the latter serves as the major binding site mediating the binding of both soluble polysaccharide and insoluble ligands. In the present study we have for the first time demonstrated that the key ligand-binding residues of RoGACBM21 can be identified and characterized by a combination of novel bioinformatics methodologies in the absence of resolved three-dimensional structural information.

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Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei

Carbohydrate Research ◽

10.1016/j.carres.2011.01.019 ◽

2011 ◽

Vol 346 (6) ◽

pp. 839-846 ◽

Cited By ~ 20

Author(s):

Letizia Tavagnacco ◽

Philip E. Mason ◽

Udo Schnupf ◽

Felicia Pitici ◽

Linghao Zhong ◽

...

Keyword(s):

Trichoderma Reesei ◽

Binding Sites ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Sugar Binding

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The Crystal Structure of the Family 6 Carbohydrate Binding Module fromCellvibrio mixtusEndoglucanase 5A in Complex with Oligosaccharides Reveals Two Distinct Binding Sites with Different Ligand Specificities

Journal of Biological Chemistry ◽

10.1074/jbc.m401599200 ◽

2004 ◽

Vol 279 (20) ◽

pp. 21560-21568 ◽

Cited By ~ 52

Author(s):

Virgínia M. R. Pires ◽

Joanna L. Henshaw ◽

José A. M. Prates ◽

David N. Bolam ◽

Luís M. A. Ferreira ◽

...

Keyword(s):

Crystal Structure ◽

Binding Sites ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

The Family

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Recombinant family 3 carbohydrate-binding module as a new additive for enhanced enzymatic saccharification of whole slurry from autohydrolyzed Eucalyptus globulus wood

Cellulose ◽

10.1007/s10570-018-1722-6 ◽

2018 ◽

Vol 25 (4) ◽

pp. 2505-2514 ◽

Cited By ~ 2

Author(s):

Carla Oliveira ◽

Aloia Romaní ◽

Daniel Gomes ◽

Joana T. Cunha ◽

Francisco M. Gama ◽

...

Keyword(s):

Eucalyptus Globulus ◽

Enzymatic Saccharification ◽

Carbohydrate Binding ◽

Carbohydrate Binding Module ◽

Whole Slurry

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Offretite Zeolite Single Crystals Synthesized by Amphiphile-Templating Approach

Molecules ◽

10.3390/molecules26082238 ◽

2021 ◽

Vol 26 (8) ◽

pp. 2238

Author(s):

Eng-Poh Ng ◽

Nur Hidayahni Ahmad ◽

Fitri Khoerunnisa ◽

Svetlana Mintova ◽

Tau Chuan Ling ◽

...

Keyword(s):

Cetyltrimethylammonium Bromide ◽

Cost Effective ◽

Acid Sites ◽

Biofuel Production ◽

Structure Directing Agent ◽

High Crystallinity ◽

Organic Templates ◽

Synthesis Approach ◽

Conventional Counterpart ◽

Dual Functions

Offretite zeolite synthesis in the presence of cetyltrimethylammonium bromide (CTABr) is reported. The offretite crystals were synthesized with a high crystallinity and hexagonal prismatic shape after only 72 h of hydrothermal treatment at 180 °C. The CTABr has dual-functions during the crystallization of offretite, viz. as structure-directing agent and as mesoporogen. The resulting offretite crystals, with a Si/Al ratio of 4.1, possess more acid sites than the conventional offretite due to their high crystallinity and hierarchical structure. The synthesized offretite is also more reactive than its conventional counterpart in the acylation of 2-methylfuran for biofuel production under non-microwave instant heating condition, giving 83.5% conversion with 100% selectivity to the desired product 2-acetyl-5-methylfuran. Hence, this amphiphile synthesis approach offers another cost-effective and alternative route for crystallizing zeolite materials that require expensive organic templates.

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Diverse Banana Pseudostems and Rachis Are Distinctive for Edible Carbohydrates and Lignocellulose Saccharification towards High Bioethanol Production under Chemical and Liquid Hot Water Pretreatments

Molecules ◽

10.3390/molecules26133870 ◽

2021 ◽

Vol 26 (13) ◽

pp. 3870

Author(s):

Jingyang Li ◽

Fei Liu ◽

Hua Yu ◽

Yuqi Li ◽

Shiguang Zhou ◽

...

Keyword(s):

Soluble Sugars ◽

Enzymatic Saccharification ◽

Hot Water ◽

Biofuel Production ◽

Bioenergy Crops ◽

Bioethanol Production ◽

Factors Affecting ◽

Liquid Hot Water ◽

Biomass Processing ◽

Banana Crops

Banana is a major fruit crop throughout the world with abundant lignocellulose in the pseudostem and rachis residues for biofuel production. In this study, we collected a total of 11 pseudostems and rachis samples that were originally derived from different genetic types and ecological locations of banana crops and then examined largely varied edible carbohydrates (soluble sugars, starch) and lignocellulose compositions. By performing chemical (H2SO4, NaOH) and liquid hot water (LHW) pretreatments, we also found a remarkable variation in biomass enzymatic saccharification and bioethanol production among all banana samples examined. Consequently, this study identified a desirable banana (Refen1, subgroup Pisang Awak) crop containing large amounts of edible carbohydrates and completely digestible lignocellulose, which could be combined to achieve the highest bioethanol yields of 31–38% (% dry matter), compared with previously reported ones in other bioenergy crops. Chemical analysis further indicated that the cellulose CrI and lignin G-monomer should be two major recalcitrant factors affecting biomass enzymatic saccharification in banana pseudostems and rachis. Therefore, this study not only examined rich edible carbohydrates for food in the banana pseudostems but also detected digestible lignocellulose for bioethanol production in rachis tissue, providing a strategy applicable for genetic breeding and biomass processing in banana crops.

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