The cellulose/lignin assembly assessed by molecular modeling. Part 2: Seeking for evidence of organization of lignin molecules at the interface with cellulose.

We have extended our previous computational investigation of the cellulose lignin assembly by considering more complex systems. Surface coverage of cellulose, structural parameters such as molecular mass and structural features of the lignin models and the presence of an explicit hydrated environment have been taken into account to examine their influence on the associative interactions between cellulose and lignin. To this end, different lignin molecular models, from beta-O-4 dimers up to a 20-units oligomer, were considered. Independently of the system studied, the key feature of the adsorption is globally preserved: aromatic rings of lignin adopt a preferential parallel orientation relative to the cellulose surface. Such structural order appears to be limited to the first shell of lignin units adsorbed on the cellulose. The pre-organization of the lignin monolayer at the surface of cellulose is not significantly changed at the interface with water. However, adsorption significantly depends on the molecular mass and the structure of lignin. The structural order is significantly hindered by the presence of branching or some particular inter-units linkages in the structure of lignin. Such results rationalize the apparent contradiction between the available experimental results.     

In-situ Raman microprobe studies of plant cell walls: Macromolecular organization and compositional variability in the secondary wall of Picea mariana (Mill.) B.S.P.

Native-state organization and distribution of cell-wall components in the secondary wall of woody tissue from P. mariana (Black Spruce) have been investigated using polarized Raman microspectroscopy. Evidence for orientation is detected through Raman intensity variations resulting from rotations of the exciting electric vector with respect to cell-wall geometry. Spectral features associated with cellulose and lignin were studied. The changes in cellulose bands indicate that the pyranose rings of the anhydroglucose repeat units are in planes perpendicular to the cross section, while methine C–H bonds are in planes parallel to the cross section. Changes in bands associated with lignin indicate that the aromatic rings of the phenyl-propane units are most often in the plane of the cell-wall surface. However, regions where lignin orientation departs from this pattern also occur. These results represent direct evidence of molecular organization with respect to cellular morphological features in woody tissue, and indicate that cell-wall components are more highly organized than had been recognized. Studies carried out in order to establish the usefulness and sensitivity of the Raman technique to differences of composition within the cell walls provide evidence of variations in the distribution of cellulose and lignin. Such compositional differences were more prominent between the walls of different cells than within a particular cell wall.     

Binding preferences, surface attachment, diffusivity, and orientation of a family 1 carbohydrate-binding module on cellulose

Cellulase enzymes often contain carbohydrate-binding modules (CBMs) for binding to cellulose. The mechanisms by which CBMs recognize specific surfaces of cellulose and aid in deconstruction are essential to understand cellulase action. The Family 1 CBM from the Trichoderma reesei Family 7 cellobiohydrolase, Cel7A, is known to selectively bind to hydrophobic surfaces of native cellulose. It is most commonly suggested that three aromatic residues identify the planar binding face of this CBM, but several recent studies have challenged this hypothesis. Here, we use molecular simulation to study the CBM binding orientation and affinity on hydrophilic and hydrophobic cellulose surfaces. Roughly 43 μs of molecular dynamics simulations were conducted, which enables statistically significant observations. We quantify the fractions of the CBMs that detach from crystal surfaces or diffuse to other surfaces, the diffusivity along the hydrophobic surface, and the overall orientation of the CBM on both hydrophobic and hydrophilic faces. The simulations demonstrate that there is a thermodynamic driving force for the Cel7A CBM to bind preferentially to the hydrophobic surface of cellulose relative to hydrophilic surfaces. In addition, the simulations demonstrate that the CBM can diffuse from hydrophilic surfaces to the hydrophobic surface, whereas the reverse transition is not observed. Lastly, our simulations suggest that the flat faces of Family 1 CBMs are the preferred binding surfaces. These results enhance our understanding of how Family 1 CBMs interact with and recognize specific cellulose surfaces and provide insights into the initial events of cellulase adsorption and diffusion on cellulose.     

Cellulose-Lignin Interactions (A Computational Study)

Within a broader program of study of the molecular structure of plant cell walls, molecular dynamics calculations were used to explore the character of the motion of lignin model compounds near a cellulose surface. Model cellulose microfibrils, which have a large number of hydroxyl groups on the surface, appear to have a net attractive interaction with the lignin models examined in this study. The lignin monomer coniferyl alcohol rapidly adsorbed onto the surface from a water layer after it was released 13 A from the surface. The major long-range force responsible for this adsorption is likely electrostatic. The attractive interaction is sufficient to restrict the motion of coniferyl alcohol when it is within 1 A of the surface and to orient the phenyl ring parallel to the surface. The [beta]-O-4-linked trimer also was observed to adsorb onto the surface with two of its phenyl rings parallel to the surface. These results suggest a mechanism by which the polysaccharide component of the plant cell wall could influence the structure of lignin. Furthermore, they provide a rationalization of the experimental observation that polysaccharides can change the course of dehydrogenation polymerization of cinnamyl alcohols.     

Equilibrium water contents of cellulose films determined via solvent exchange and quartz crystal microbalance with dissipation monitoring

Model cellulose surfaces have attracted increasing attention for studying interactions with cell wall matrix polymers and as substrates for enzymatic degradation studies. Quartz crystal microbalance with dissipation monitoring (QCM-D) solvent exchange studies showed that the water content of regenerated cellulose (RC) films was proportional to the film thickness (d) and was consistent with about five water molecules per anhydroglucose unit. Sulfated nanocrystalline cellulose (SNC) and desulfated nanocrystalline cellulose (DNC) films had comparable water contents and contained about five times more water than RC films. A cellulase mixture served as a probe for studies of substrate accessibility and degradation. Cellulase adsorption onto RC films was independent of d, whereas degradation times increased with d. However, adsorption onto SNC and DNC films increased with d, whereas cellulase degradation times for DNC films were independent of studied d. Enhanced access to guest molecules for SNC and DNC films revealed they are more porous than RC films.     

Seeing double: Crystal structures of the type I TNF receptor

The crystal structure of the extracellular domain of the type I tumor necrosis factor receptor (sTNF-R1) has been determined to 2.25 Å at pH 7.5. We have also solved the structure of sTNF-R1 at pH 3.7. sTNF-R1 is an elongated molecule consisting of a linear combination of four cysteine-rich motifs. Interestingly, the crystal structure reveals two distinct dimers of the receptor. One dimer is formed by a parallel arrangement of receptors, the other by an antiparallel arrangement of receptors. In the parallel arrangement of the receptors, the tumor necrosis factor (TNF) binding face of the receptor is completely exposed to solvent. However, in the antiparallel arrangement, the TNF binding face is intimately involved in the dimer interactions. Details of these recognition surfaces are discussed. Both these dimer interactions bury substantial surface area, comprise polar and apolar contact surfaces and have complimentary recognition surfaces. Thus these interactions are typical of genuine protein–protein interactions, rather than crystal packing contacts. These dimers may function to inhibit signal transduction in the absence of TNF or in the case of the parallel dimer, promote clustering of TNF/TNF receptor complexes on the cell surface.     

The adsorption of xyloglucan on cellulose: effects of explicit water and side chain variation

The interaction between para-crystalline cellulose and the cross-linking glycan xyloglucan (XG) plays a central role for the strength and extensibility of plant cell walls. The coating of XGs on cellulose surfaces is believed to be one of the most probable interaction patterns. In this work, the effects of explicit water and side chain variation on the adsorption of XGs on cellulose are investigated by means of atomistic molecular dynamics simulations. The adsorption properties are studied in detail for three XGs on cellulose Iβ 1–10 surface in aqueous environment, namely GXXXGXXXG, GXXLGXXXG, and GXXFGXXXG, which differ in the length and composition of one side chain. Our work shows that when water molecules are included in the theoretical model, the total interaction energies between the adsorbed XGs and cellulose are considerably smaller than in vacuo. Furthermore, in water environment the van der Waals interactions prevail over the electrostatic interactions in the adsorption. Variation in one side chain does not have significant influence on the interaction energy and the binding affinity, but does affect the equilibrium structural properties of the adsorbed XGs to facilitate the interaction between both the backbone and the side chain residues with the cellulose surface. Together, this analysis provides new insights into the nature of the XG–cellulose interaction, which helps to further refine current molecular models of the composite plant cell wall.     

Predicting Enzyme Adsorption to Lignin Films by Calculating Enzyme Surface Hydrophobicity

The inhibitory action of lignin on cellulase cocktails is a major challenge to the biological saccharification of plant cell wall polysaccharides. Although the mechanism remains unclear, hydrophobic interactions between enzymes and lignin are hypothesized to drive adsorption. Here we evaluate the role of hydrophobic interactions in enzyme-lignin binding. The hydrophobicity of the enzyme surface was quantified using an estimation of the clustering of nonpolar atoms, identifying potential interaction sites. The adsorption of enzymes to lignin surfaces, measured using the quartz crystal microbalance, correlates to the hydrophobic cluster scores. Further, these results suggest a minimum hydrophobic cluster size for a protein to preferentially adsorb to lignin. The impact of electrostatic contribution was ruled out by comparing the isoelectric point (pI) values to the adsorption of proteins to lignin surfaces. These results demonstrate the ability to predict enzyme-lignin adsorption and could potentially be used to design improved cellulase cocktails, thus lowering the overall cost of biofuel production.     

Cellulase–lignin interactions—The role of carbohydrate-binding module and pH in non-productive binding

Non-productive cellulase adsorption onto lignin is a major inhibitory mechanism preventing enzymatic hydrolysis of lignocellulosic feedstocks. Therefore, understanding of enzyme–lignin interactions is essential for the development of enzyme mixtures and processes for lignocellulose hydrolysis. We have studied cellulase–lignin interactions using model enzymes, Melanocarpus albomyces Cel45A endoglucanase (MaCel45A) and its fusions with native and mutated carbohydrate-binding modules (CBMs) from Trichoderma reesei Cel7A. Binding of MaCel45A to lignin was dependent on pH in the presence and absence of the CBM; at high pH, less enzyme bound to isolated lignins. Potentiometric titration of the lignin preparations showed that negatively charged groups were present in the lignin samples and that negative charge in the samples was increased with increasing pH. The results suggest that electrostatic interactions contributed to non-productive enzyme adsorption: Reduced enzyme binding at high pH was presumably due to repulsive electrostatic interactions between the enzymes and lignin. The CBM increased binding of MaCel45A to the isolated lignins only at high pH. Hydrophobic interactions are probably involved in CBM binding to lignin, because the same aromatic amino acids that are essential in CBM–cellulose interaction were also shown to contribute to lignin-binding.     

Energetics for displacing a single chain from the surface of microcrystalline cellulose into the active site of Acidothermus cellulolyticus Cel5A

A series of molecular mechanics calculations were used to analyze the energetics for moving a single polysaccharide chain from the surface of microcrystalline cellulose into the binding cleft of the Cel5A cellulase from Acidothermus cellulolyticus. A build-up procedure was used to model the placement of a 12-residue oligosaccharide chain along the surface of the enzyme, using as a guide the four residues of the tetrasaccharide substrate co-crystallized with the protein in the crystallographic structure determination. The position of this 12-residue oligosaccharide was used to orient the enzyme properly above two different surfaces of cellulose 1beta, the (1,0,0) and the (1,1,0) faces of the crystal. Constrained molecular dynamics simulations were then used to pull a target chain directly below the enzyme up out of the crystal surface and into the binding groove. The energetics for this process were favorable for both faces, with the step face being more favorable than the planar face, implying that this surface could be hydrolyzed more readily.     

Reduced cellulose synthesis invokes lignification and defense responses in Arabidopsis thaliana

The cell wall determines the shape of plant cells and is also the primary interface for pathogen interactions. The structure of the cell wall can be modified in response to developmental and environmental cues, for example to strengthen the wall and to create barriers to pathogen ingress. The ectopic lignin 1-1 and 1-2 (eli1-1 and eli1-2) mutations lead to an aberrant deposition of lignin, a complex phenylpropanoid polymer. We show that the eli1 mutants occur in the cellulose synthase gene CESA3 in Arabidopsis thaliana and cause reduced cellulose synthesis, providing further evidence for the function of multiple CESA subunits in cellulose synthesis. We show that reduced levels of cellulose synthesis, caused by mutations in cellulose synthase genes and in genes affecting cell expansion, activate lignin synthesis and defense responses through jasmonate and ethylene and other signaling pathways. These observations suggest that mechanisms monitoring cell wall integrity can activate lignification and defense responses.     

Model films from native cellulose nanofibrils. Preparation, swelling, and surface interactions

Native cellulose model films containing both amorphous and crystalline cellulose I regions were prepared by spin-coating aqueous cellulose nanofibril dispersions onto silica substrates. Nanofibrils from wood pulp with low and high charge density were used to prepare the model films. Because the low charged nanofibrils did not fully cover the silica substrates, an anchoring substance was selected to improve the coverage. The model surfaces were characterized using atomic force microscopy (AFM) and X-ray photoelectron spectroscopy (XPS). The effect of nanofibril charge density, electrolyte concentration, and pH on swelling and surface interactions of the model film was studied by quartz crystal microbalance with dissipation (QCM-D) and AFM force measurements. The results showed that the best coverage for the low charged fibrils was achieved by using 3-aminopropyltrimethoxysilane (APTS) as an anchoring substance and hence it was chosen as the anchor. The AFM and XPS measurements showed that the fibrils are covering the substrates. Charge density of the fibrils affected the morphology of the model surfaces. The low charged fibrils formed a network structure while the highly charged fibrils formed denser film structure. The average thickness of the films corresponded to a monolayer of fibrils, and the average rms roughness of the films was 4 and 2 nm for the low and high charged nanofibril films, respectively. The model surfaces were stable in QCM-D swelling experiments, and the behavior of the nanofibril surfaces at different electrolyte concentrations and pHs correlated with other studies and the theories of Donnan. The AFM force measurements with the model surfaces showed well reproducible results, and the swelling results correlated with the swelling observed by QCM-D. Both steric and electrostatic forces were observed and the influence of steric forces increased as the films were swelling due to changes in pH and electrolyte concentration. These films differ from previous model cellulose films due to their chemical composition (crystalline cellulose I and amorphous regions) and fibrillar structure and hence serve as excellent models for the pulp fiber surface.     

Impact of lignins isolated from pretreated lignocelluloses on enzymatic cellulose saccharification

Lignins were enzymatically isolated from corn stover and wheat straw samples and subjected to hydrothermal or wet oxidation pretreatments for enzyme adsorption experimentations. Lignin contents of the isolates ranged from 26 to 71 % (w/w); cellulose ranged from 3 to 22 % (w/w); xylan from 0.7 to 6 % (w/w) and ash was from 5.8 to 30 % (w/w). ATR-IR analyses indicated significant and similar levels of calcium in all lignin isolates. Commercial cellulase adsorption studies showed that the presence of these lignins had no significant impact on the total amount of adsorbed enzyme in cellulose and cellulose–lignin systems. Consequently, the presence of the lignins had minimal effect, if any, on enzymatic cellulose conversion. Furthermore, this result, coupled with significant calcium levels in the isolated lignins, supports previous work suggesting lignin–calcium complexes reduce enzyme–lignin interactions.     

A Raman-scattering study on the net orientation of biomacromolecules in the outer epidermal walls of mature wheat stems (Triticum aestivum)

BACKGROUND AND AIMS: Raman spectroscopy can be used to examine the orientation of biomacromolecules using relatively thick samples of material, whereas more traditional means of analysing molecular structure require prior isolation of the components, which often destroys morphological features. In this study, Raman spectroscopy was used to examine the outer epidermal cell walls of wheat stems. METHODS: Polarized Raman spectra from the epidermal cell walls of wheat stem were obtained using near-infrared-Fourier transform Raman scattering. By comparing spectra taken with Raman light polarized perpendicular or parallel to the longitudinal axis of the cell, the orientation of macromolecules in the cell wall was investigated. KEY RESULTS: The net orientation of macromolecules varies in the epidermal cell walls of the different components of wheat stem. The net orientation of cellulose is parallel to the longitudinal axis of the cells, whereas the xylan and the phenylpropane units of lignin tend to lie perpendicular to the longitudinal axis of the cells, i.e. perpendicular to the net orientation of cellulose in the epidermal cell walls. CONCLUSIONS: The results imply that cellulose, lignin and xylan form a relatively ordered network that defines the mechanical and structural properties of the cell wall. Such results are likely to have a significant impact on the formulation of definitive models for the static and growing cell wall.     

Structural Insights into the Affinity of Cel7A Carbohydrate-binding Module for Lignin

The high cost of hydrolytic enzymes impedes the commercial production of lignocellulosic biofuels. High enzyme loadings are required in part due to their non-productive adsorption to lignin, a major component of biomass. Despite numerous studies documenting cellulase adsorption to lignin, few attempts have been made to engineer enzymes to reduce lignin binding. In this work, we used alanine-scanning mutagenesis to elucidate the structural basis for the lignin affinity of Trichoderma reesei Cel7A carbohydrate binding module (CBM). T. reesei Cel7A CBM mutants were produced with a Talaromyces emersonii Cel7A catalytic domain and screened for their binding to cellulose and lignin. Mutation of aromatic and polar residues on the planar face of the CBM greatly decreased binding to both cellulose and lignin, supporting the hypothesis that the cellulose-binding face is also responsible for lignin affinity. Cellulose and lignin affinity of the 31 mutants were highly correlated, although several mutants displayed selective reductions in lignin or cellulose affinity. Four mutants with increased cellulose selectivity (Q2A, H4A, V18A, and P30A) did not exhibit improved hydrolysis of cellulose in the presence of lignin. Further reduction in lignin affinity while maintaining a high level of cellulose affinity is thus necessary to generate an enzyme with improved hydrolysis capability. This work provides insights into the structural underpinnings of lignin affinity, identifies residues amenable to mutation without compromising cellulose affinity, and informs engineering strategies for family one CBMs.     

Towards Biomimicking Wood: Fabricated Free-standing Films of Nanocellulose,Lignin, and a Synthetic Polycation

Woody materials are comprised of plant cell walls that contain a layered secondary cell wall composed of structural polymers of polysaccharides and lignin. Layer-by-layer (LbL) assembly process which relies on the assembly of oppositely charged molecules from aqueous solutions was used to build a freestanding composite film of isolated wood polymers of lignin and oxidized nanofibril cellulose (NFC). To facilitate the assembly of these negatively charged polymers, a positively charged polyelectrolyte, poly(diallyldimethylammomium chloride) (PDDA), was used as a linking layer to create this simplified model cell wall. The layered adsorption process was studied quantitatively using quartz crystal microbalance with dissipation monitoring (QCM-D) and ellipsometry. The results showed that layer mass/thickness per adsorbed layer increased as a function of total number of layers. The surface coverage of the adsorbed layers was studied with atomic force microscopy (AFM). Complete coverage of the surface with lignin in all the deposition cycles was found for the system, however, surface coverage by NFC increased with the number of layers. The adsorption process was carried out for 250 cycles (500 bilayers) on a cellulose acetate (CA) substrate. Transparent free-standing LBL assembled nanocomposite films were obtained when the CA substrate was later dissolved in acetone. Scanning electron microscopy (SEM) of the fractured cross-sections showed a lamellar structure, and the thickness per adsorption cycle (PDDA-Lignin-PDDA-NC) was estimated to be 17 nm for two different lignin types used in the study. The data indicates a film with highly controlled architecture where nanocellulose and lignin are spatially deposited on the nanoscale (a polymer-polymer nanocomposites), similar to what is observed in the native cell wall.     

Engineering and characterization of carbohydrate-binding modules for imaging cellulose fibrils biosynthesis in plant protoplasts

Carbohydrate binding modules (CBMs) are noncatalytic domains that assist tethered catalytic domains in substrate targeting. CBMs have therefore been used to visualize distinct polysaccharides present in the cell wall of plant cells and tissues. However, most previous studies provide a qualitative analysis of CBM-polysaccharide interactions, with limited characterization of engineered tandem CBM designs for recognizing polysaccharides like cellulose and limited application of CBM-based probes to visualize cellulose fibrils synthesis in model plant protoplasts with regenerating cell walls. Here, we examine the dynamic interactions of engineered type-A CBMs from families 3a and 64 with crystalline cellulose-I and phosphoric acid swollen cellulose. We generated tandem CBM designs to determine various characteristic properties including binding reversibility toward cellulose-I using equilibrium binding assays. To compute the adsorption (nk_on) and desorption (k_off) rate constants of single versus tandem CBM designs toward nanocrystalline cellulose, we employed dynamic kinetic binding assays using quartz crystal microbalance with dissipation. Our results indicate that tandem CBM3a exhibited the highest adsorption rate to cellulose and displayed reversible binding to both crystalline/amorphous cellulose, unlike other CBM designs, making tandem CBM3a better suited for live plant cell wall biosynthesis imaging applications. We used several engineered CBMs to visualize Arabidopsis thaliana protoplasts with regenerated cell walls using confocal laser scanning microscopy and wide-field fluorescence microscopy. Lastly, we also demonstrated how CBMs as probe reagents can enable in situ visualization of cellulose fibrils during cell wall regeneration in Arabidopsis protoplasts.     

Equilibria for the adsorption of antibiotics onto neutral polymeric sorbents: Experimental and modeling studies

The objective of this work was to study the equilibria for adsorption of three antibiotics (penicillin V, tetracycline, and cephalosporin C) from water onto commercially available neutral polymeric sorbents. The pH was observed to be an important factor in adsorption as our results suggest that the neutral forms of penicillin V and cephalosporin C are preferentially adsorbed onto the neutral sorbents. Also, sorbent surface chemistry was observed to be important for adsorption, as the antibiotics adsorbed more favorably (both in terms of affinities and enthalpies) onto the aromatic sorbent as compared to the aliphatic ester sorbent. In addition to these thermodynamic measurements, molecular modeling studies and Monte Carlo simulations suggest that adsorption onto aromatic sorbents may involve specific interactions between the planar regions of the antibiotic molecules and the phenyl rings of the aromatic sorbent. The interaction energies predicted from Monte Carlo simulations were observed to provide qualitative agreement with experimentally determined adsorption affinities. (c) 1995 John Wiley & Sons, Inc.     

Adsorption of a xylanase purified from Pulpzyme HC onto alkali-lignin and crystalline cellulose

Adsorption of a xylanase purified from a commercial xylanase, Pulpzyme HC, onto two model components of kraft pulp, crystalline cellulose (Avicel) and alkali-lignin (Indulin AT), was studied at 40°C. A considerable amount of the purified xylanase was adsorbed onto alkali-lignin in alkaline solutions. The adsorption of the purified xylanase onto crystalline cellulose was not significant and could be described by the Langmuir-type adsorption isotherm. The adsorption of the purified xylanase onto alkali-lignin was assumed to be caused by physical or van der Waals interaction based on the result that NaCl did not change the adsorption isotherm. © Rapid Science Ltd. 1998     

Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose.

The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution. The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion. conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule. One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose. The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs. On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented. Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface. The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains. In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.