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Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that oxidatively cleave recalcitrant polysaccharides such as cellulose. Several studies have reported LPMO action in synergy with other carbohydrate-active enzymes (CAZymes) for the degradation of lignocellulosic biomass but direct LPMO action at the plant tissue level remains challenging to investigate. Here, we have developed a MALDI-MS imaging workflow to detect oxidised oligosaccharides released by a cellulose-active LPMO at cellular level on maize tissues. Using this workflow, we imaged LPMO action and gained insight into the spatial variation and relative abundance of oxidised and non-oxidised oligosaccharides. We reveal a targeted action of the LPMO related to the composition and organisation of plant cell walls., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

Understanding lignocellulosic biomass resistance to enzymatic deconstruction is crucial for its sustainable conversion into bioproducts. Despite scientific advances, quantitative morphological analysis of plant deconstruction at cell and tissue scales remains under-explored. In this study, an original pipeline is devised, involving four-dimensional (space  + time) fluorescence confocal imaging, and a novel computational tool, to track and quantify deconstruction at cell and tissue scales. By applying this pipeline to poplar wood, dynamics of cellular parameters was computed and cellulose conversion during enzymatic deconstruction was measured. Results showed that enzymatic deconstruction predominantly impacts cell wall volume rather than surface area. Additionally, a negative correlation was observed between pre-hydrolysis compactness measures and volumetric cell wall deconstruction rate, whose strength was modulated by enzymatic activity. Results also revealed a strong positive correlation between average volumetric cell wall deconstruction rate and cellulose conversion rate. These findings link key deconstruction parameters across nano and micro scales., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The diversity of the plant biomass available on earth makes plants an exceptional resource for replacing fossil resources in green chemistry, bioenergy and biobased materials. For numerous applications, and especially the high-tech ones (building block molecules, high-power bioenergy, additive manufacturing of biobased materials), the macrostructure assemblies of the plant biomass often need to be first broken down into a fine powder. This can be achieved by dry fractionation process combining comminution and sorting steps. The chemical and physical properties of the ground plant powder results both from the process conditions, the histological structure and chemical composition of the raw plant materials. In a forward engineering approach, the quality of the final products can be highly improved by the selection of the right powder (raw materials and production process) for the right application. This article provides production routes together with physical and chemical characterization of 10 biomass powders from 6 different biomass feedstocks (SP - spirulina, HI - hibiscus, PB - pine bark, HC - hemp Core, RH - rice husk and RHA - rice husk ash). These feedstocks represent a broad range of raw materials properties. For pine bark, hemp core, rice husk and rice husk ash, two grades of powders related to two different particle sizes were produced by two different routes to highlight the impact of the comminution process on the powder properties. The devices used and the process parameters are described. The morphological properties of the powder were quantified using laser diffraction (particle size) and image analysis (shape factor) and qualitatively analyzed with SEM. The specific surface area was determined using gas sorption with BET theory, and the hygroscopic properties were measured using direct vapor sorption. The chemical characterizations were determined with a set of biochemical assays and, complementary, FTIR and fluorescence spectra were recorded to provide fingerprints of samples. The dataset includes tables that summarize the main characteristic descriptors of each analysis as well as the raw data. The data are registered in the French Research Data Gouv public repository and also stored in the PO2 BaGaTel database using the PO2/TransformON ontology [1]. SPO2Q web tool allows on line querying of the database, which can also be consulted using PO2 manager desktop application [[1], [2], [3]]., (© 2024 The Author(s).)

Background: Lignocellulosic biomass is a complex network of polysaccharides and lignin that requires a pretreatment step to overcome recalcitrance and optimize valorisation into biobased products. Pretreatment of biomass induces chemical and morphological changes. Quantification of these changes is critical to understand biomass recalcitrance and to predict lignocellulose reactivity. In this study, we propose an automated method for the quantification of chemical and morphological parameters through fluorescence macroscopy, which was applied on wood samples (spruce, beechwood) pretreated with steam explosion., Results: Results in fluorescence macroscopy highlighted the impact of steam explosion on spruce and beechwood: fluorescence intensity of samples was highly altered, especially for the most severe conditions. Morphological changes were also revealed: shrinkage of cells and deformation of cell walls manifested as the loss of rectangularity or circular shape, for tracheids in spruce and vessels in beechwood respectively. Quantification of fluorescence intensity of cell walls and quantification of morphological parameters related to cell lumens were carried out accurately by applying the automated method onto the macroscopic images. The results showed that lumens area and circularity could be considered as complementary markers of cell deformation, and that fluorescence intensity of the cell walls could be related to morphological changes and to the conditions of pretreatment., Conclusions: The developed procedure allows simultaneous and effective quantification of morphological parameters and fluorescence intensity of the cell walls. This approach can be applied to fluorescence macroscopy as well as other imaging techniques and provides encouraging results towards the understanding of biomass architecture., (© 2023. The Author(s).)

This review provides a critical analysis of the state of the art of dilute acid pretreatment applied to lignocellulosic biomass. Data from 63 publications were extracted and analysed. The majority of the papers used residence times of<30 min, temperature ranges from 100 °C to 200 °C, and acid levels between 0 % and 2 %. Yields are quantified directly after pretreatment (xylose content) or after enzymatic hydrolysis (glucose content). Statistical analyses allowed the time-temperature equivalence to be quantified for three types of biomass: they were formulated by non-linear expressions. In further works, investigating less explored areas, for example moderate temperature levels with longer residence times, is recommended. Pretreatment material (time-temperature kinetics, reactor type) and analytical methods should be standardized and better described. It becomes mandatory to promote the development of an open, findable, accessible, interoperable, and reusable data approach for pretreatments research., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The interactions of proteins involved in plant cell wall hydrolysis, such as enzymes and CBMs, significantly determine their role and efficiency. In order to go beyond the characterization of interactions with simple ligands, bioinspired assemblies combined with the measurement of diffusion and interaction by FRAP offer a relevant alternative for highlighting the importance of different parameters related to the protein affinity and to the polymer type and organization in the assembly., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

This work presents a dynamic view of the enzymatic degradation of maize cell walls, and sheds new light on the recalcitrance of hot water pretreated maize stem internodes. Infra-red microspectrometry, mass spectrometry, fluorescence recovery after photobleaching and fluorescence imaging were combined to investigate enzymatic hydrolysis at the cell scale. Depending on their polymer composition and organisation, cell types exhibits different extent and rate of enzymatic degradation. Enzymes act sequentially from the cell walls rich in accessible cellulose to the most recalcitrant cells. This phenomenon can be linked to the heterogeneous distribution of enzymes in the liquid medium and the adsorption/desorption mechanisms that differ with the type of cell., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Background: Biomass recalcitrance is governed by various molecular and structural factors but the interplay between these multiscale factors remains unclear. In this study, hot water pretreatment (HWP) was applied to maize stem internodes to highlight the impact of the ultrastructure of the polymers and their interactions on the accessibility and recalcitrance of the lignocellulosic biomass. The impact of HWP was analysed at different scales, from the polymer ultrastructure or water mobility to the cell wall organisation by combining complementary compositional, spectral and NMR analyses., Results: HWP increased the kinetics and yield of saccharification. Chemical characterisation showed that HWP altered cell wall composition with a loss of hemicelluloses (up to 45% in the 40-min HWP) and of ferulic acid cross-linking associated with lignin enrichment. The lignin structure was also altered (up to 35% reduction in β-O-4 bonds), associated with slight depolymerisation/repolymerisation depending on the length of treatment. The increase in [Formula: see text], [Formula: see text] and specific surface area (SSA) showed that the cellulose environment was looser after pretreatment. These changes were linked to the increased accessibility of more constrained water to the cellulose in the 5-15 nm pore size range., Conclusion: The loss of hemicelluloses and changes in polymer structural features caused by HWP led to reorganisation of the lignocellulose matrix. These modifications increased the SSA and redistributed the water thereby increasing the accessibility of cellulases and enhancing hydrolysis. Interestingly, lignin content did not have a negative impact on enzymatic hydrolysis but a higher lignin condensed state appeared to promote saccharification. The environment and organisation of lignin is thus more important than its concentration in explaining cellulose accessibility. Elucidating the interactions between polymers is the key to understanding LB recalcitrance and to identifying the best severity conditions to optimise HWP in sustainable biorefineries., (© 2021. The Author(s).)

Enzymes are essential and ubiquitous biocatalysts involved in various metabolic pathways and used in many industrial processes. Here, we reframe enzymes not just as biocatalysts transforming bioproducts but also as sensitive probes for exploring the structure and composition of complex bioproducts, like meat tissue, dairy products and plant materials, in both food and non-food bioprocesses. This review details the global strategy and presents the most recent investigations to prepare and use enzymes as relevant probes, with a focus on glycoside-hydrolases involved in plant deconstruction and proteases and lipases involved in food digestion. First, to expand the enzyme repertoire to fit bioproduct complexity, novel enzymes are mined from biodiversity and can be artificially engineered. Enzymes are further characterized by exploring sequence/structure/dynamics/function relationships together with the environmental factors influencing enzyme interactions with their substrates. Then, the most advanced experimental and theoretical approaches developed for exploring bioproducts at various scales (from nanometer to millimeter) using active and inactive enzymes as probes are illustrated. Overall, combining multimodal and multiscale approaches brings a better understanding of native-form or transformed bioproduct architecture and composition, and paves the way to mainstream the use of enzymes as probes., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

In lignocellulosic biomass (LB), the activity of enzymes is limited by the appearance of non-specific interactions with lignin during the hydrolysis process, which maintains enzymes far from their substrate. Characterization of these complex interactions is thus a challenge in complex substrates such as LB. The method here measures molecular interactions between fluorophore-tagged molecules and native autofluorescent lignin, to be revealed by Förster resonance energy transfer (FRET). Contrary to FRET measurements in living cells using two exogenous fluorophores, FRET measurements in plants using lignin is not trivial due to its complex autofluorescence. We have developed an original acquisition and analysis pipeline with correlated observation of two complementary properties of fluorescence: fluorescence emission and lifetime. sFLIM (spectral and fluorescent lifetime imaging microscopy) provides the quantification of these interactions with high sensitivity, revealing different interaction levels between biomolecules and lignin.

Lignocellulosic biomass (LB) is an abundant and renewable resource from plants mainly composed of polysaccharides (cellulose and hemicelluloses) and an aromatic polymer (lignin). LB has a high potential as an alternative to fossil resources to produce second-generation biofuels and biosourced chemicals and materials without compromising global food security. One of the major limitations to LB valorisation is its recalcitrance to enzymatic hydrolysis caused by the heterogeneous multi-scale structure of plant cell walls. Factors affecting LB recalcitrance are strongly interconnected and difficult to dissociate. They can be divided into structural factors (cellulose specific surface area, cellulose crystallinity, degree of polymerization, pore size and volume) and chemical factors (composition and content in lignin, hemicelluloses, acetyl groups). Goal of this review is to propose an up-to-date survey of the relative impact of chemical and structural factors on biomass recalcitrance and of the most advanced techniques to evaluate these factors. Also, recent spectral and water-related measurements accurately predicting hydrolysis are presented. Overall, combination of relevant factors and specific measurements gathering simultaneously structural and chemical information should help to develop robust and efficient LB conversion processes into bioproducts., (Copyright © 2019 Zoghlami and Paës.)

Lignocellulose biomass can be transformed into sustainable chemicals, materials and energy but its natural recalcitrance requires the use of pretreatment to enhance subsequent catalytic steps. Dilute acid pretreatment is one of the most common and efficient ones, however its impact has not yet been investigated simultaneously at nano- and cellular-scales. Poplar samples have been pretreated by dilute acid at different controlled severities, then characterized by combined structural and spectral techniques (scanning electron microscopy, confocal microscopy, autofluorescence, fluorescence lifetime, Raman). Results show that pretreatment favours lignin depolymerization until severity of 2.4-2.5 while at severity of 2.7 lignin seems to repolymerize as revealed by broadening of autofluorescence spectrum and strong decrease in fluorescence lifetime. Importantly, both nano-scale and cellular-scale markers can predict hydrolysis yield of pretreated samples, highlighting some connections in the multiscale recalcitrance of lignocellulose., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: Lignocellulose biomass is known as a recalcitrant material towards enzymatic hydrolysis, increasing the process cost in biorefinery. In nature, filamentous fungi naturally degrade lignocellulose, using an arsenal of hydrolytic and oxidative enzymes. Assessment of enzyme hydrolysis efficiency generally relies on the yield of glucose for a given biomass. To better understand the markers governing recalcitrance to enzymatic degradation, there is a need to enlarge the set of parameters followed during deconstruction., Results: Industrially-pretreated biomass feedstocks from wheat straw, miscanthus and poplar were sequentially hydrolysed following two steps. First, standard secretome from Trichoderma reesei was used to maximize cellulose hydrolysis, producing three recalcitrant lignin-enriched solid substrates. Then fungal secretomes from three basidiomycete saprotrophs ( Laetisaria arvalis, Artolenzites elegans and Trametes ljubarskyi ) displaying various hydrolytic and oxidative enzymatic profiles were applied to these recalcitrant substrates, and compared to the T. reesei secretome. As a result, most of the glucose was released after the first hydrolysis step. After the second hydrolysis step, half of the remaining glucose amount was released. Overall, glucose yield after the two sequential hydrolyses was more dependent on the biomass source than on the fungal secretomes enzymatic profile. Solid residues obtained after the two hydrolysis steps were characterized using complementary methodologies. Correlation analysis of several physico-chemical parameters showed that released glucose yield was negatively correlated with lignin content and cellulose crystallinity while positively correlated with xylose content and water sorption. Water sorption appears as a pivotal marker of the recalcitrance as it reflects chemical and structural properties of lignocellulosic biomass., Conclusions: Fungal secretomes applied to highly recalcitrant biomass samples can further extend the release of the remaining glucose. The glucose yield can be correlated to chemical and physical markers, which appear to be independent from the biomass type and secretome. Overall, correlations between these markers reveal how nano-scale properties (polymer content and organization) influence macro-scale properties (particle size and water sorption). Further systematic assessment of these markers during enzymatic degradation will foster the development of novel cocktails to unlock the degradation of lignocellulose biomass., Competing Interests: The authors declare that they have no competing interests.

Lignocellulosic biomass is considered as a sustainable source of energy and chemicals, but its recalcitrance to bioconversion still limits its use. In this paper, a strategy based on two aspects was developed to improve our knowledge on the lignin recalcitrance to enzymatic hydrolysis. First, lignocellulosic films of cellulose nanofibrils (CNFs) with increasing content of lignin (up to 40%) were prepared. Thanks to in situ real time Atomic Force Microscopy (AFM) measurements during the hydrolysis and by comparison with biochemical assays, the use of such films allows to fully assess the importance of the lignin content and of the arrangement between CNFs and lignin on the hydrolysis efficiency. In a second time, contrary to other studies by AFM which only followed a specific structure during enzymatic processes mostly on simple systems (CNFs or cellulose nanocrystals), a quantitative analysis of in-situ time-lapse measurements was developed. It enables to accurately address lignocellulosic biomass recalcitrance mechanisms mediated by lignin at nanoscale. Such analysis could pave the way for the use of a quantitative criteria to visualize in situ deconstruction of complex lignocellulosic substrates. Coupling the use of lignocellulosic films and dynamical AFM quantitative analysis to follow the evolution of the structure at nanoscale might lead to an effective targeting of new promising bioconversion strategies.

Fluorescence is a versatile property of many molecules called fluorophores. In plant cell walls, fluorescence is generally attributed to aromatic molecules such as lignin. In contrary to fluorescence intensity, fluorescence lifetime is independent from fluorophore concentration. So mapping fluorescence lifetime of plant cell walls represents a complementary approach to acquire chemical and structural information of cell wall components and interactions.

Background: Improving lignocellulolytic enzymes' diffusion and accessibility to their substrate in the plant cell walls is recognised as a critical issue for optimising saccharification. Although many chemical features are considered as detrimental to saccharification, enzymes' dynamics within the cell walls remains poorly explored and understood. To address this issue, poplar fragments were submitted to hot water and ionic liquid pretreatments selected for their contrasted effects on both the structure and composition of lignocellulose. In addition to chemical composition and porosity analyses, the diffusion of polyethylene glycol probes of different sizes was measured at three different time points during the saccharification., Results: Probes' diffusion was mainly affected by probes size and pretreatments but only slightly by saccharification time. This means that, despite the removal of polysaccharides during saccharification, diffusion of probes was not improved since they became hindered by changes in lignin conformation, whose relative amount increased over time. Porosity measurements showed that probes' diffusion was highly correlated with the amount of pores having a diameter at least five times the size of the probes. Testing the relationship with saccharification demonstrated that accessibility of 1.3-1.7-nm radius probes measured by FRAP on non-hydrolysed samples was highly correlated with poplar digestibility together with the measurement of initial porosity on the range 5-20 nm., Conclusion: Mobility measurements performed before hydrolysis can serve to explain and even predict saccharification with accuracy. The discrepancy observed between probes' size and pores' diameters to explain accessibility is likely due to biomass features such as lignin content and composition that prevent probes' diffusion through non-specific interactions probably leading to pores' entanglements.

Background: Lignocellulosic biomass is a complex network of polymers making the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that necessitates some pretreatments and several types of catalysts to be transformed efficiently. In particular, enzymes degrading lignocellulose can become inactivated due to their binding to lignin through non-specific interactions, leading to a loss in catalytic efficiency of industrial processes. Gaining more knowledge in the strength of interactions would allow optimizing enzymes and selecting appropriate pretreatments., Results: Measuring interactions directly in plant cell wall can theoretically be performed using confocal fluorescence techniques by evaluating fluorescence resonance energy transfer (FRET) between compatible fluorophores. In this study, autofluorescence of plant cell wall, mainly originating from lignin, was considered as a donor fluorophore while the acceptor was a common rhodamine-based fluorescent probe. To overcome complex plant cell wall fluorescence, which limits FRET analysis by standard techniques, we have developed an original approach, combining spectral and lifetime measurements. It consists in (1) dissecting autofluorescence signal in each spectral channel, (2) optimizing spectral channel choice for lifetime measurements and (3) achieving an unambiguous FRET signature with an autofluorescent donor fluorophore. Interactions between rhodamine-based probes of various sizes and untreated or pretreated wheat sample were evaluated, showing it was possible to discriminate interactions at the nano-scale, revealing some accessibility differences and the effect of pretreatment., Conclusions: SLiM measurement allows precise estimation of the optimal spectral range for FRET measurement. SLiM response allows for the first time doubtless FRET measurements between lignin as a donor, and an acceptor fluorophore with high accuracy and sensitivity related to lifetime decrease studies. As demonstrated, it thus becomes possible to measure interactions of fluorescent probes directly inside plant cell wall samples. This approach can thus be applied to various fields such as lignocellulose deconstruction to optimize the action of enzymes or plant cell wall development to assay in situ the biosynthesis of lignin.

Background: Biomass recalcitrance to enzymatic hydrolysis has been assigned to several structural and chemical factors. However, their relative importance remains challenging to evaluate. Three representative biomass species (wheat straw, poplar and miscanthus) were submitted to four standard pretreatments (dilute acid, hot water, ionic liquid and sodium chlorite) in order to generate a set of contrasted samples. A large array of techniques, including wet chemistry analysis, porosity measurements using NMR spectroscopy, electron and fluorescence microscopy, were used in order to determine possible generic factors of biomass recalcitrance., Results: The pretreatment conditions selected allowed obtaining samples displaying different susceptibility to enzymatic hydrolysis (from 3 up to 98% of the initial glucose content released after 96 h of saccharification). Generic correlation coefficients were calculated between the measured chemical and structural features and the final saccharification rates. Increases in porosity displayed overall strong positive correlations with saccharification efficiency, but different porosity ranges were concerned depending on the considered biomass. Lignin-related factors displayed highly negative coefficients for all biomasses. Lignin content, which is likely involved in the correlations observed for porosity, was less detrimental to enzymatic hydrolysis than lignin composition. Lignin influence was highlighted by the strong negative correlation with fluorescence intensity which mainly originates from monolignols in mature tissues., Conclusions: Our results provide a better understanding of the factors responsible for biomass recalcitrance that can reasonably be considered as generic. The correlations with specific porosity ranges are biomass species-dependent, meaning that enzymes cocktails with fitted enzyme size are likely to be needed to optimise saccharification depending on the biomass origin. Lignin composition, which probably influences its structure, is the most important parameter to overcome to enhance enzymes access to the polysaccharides. Accordingly, fluorescence intensity was found to be a rapid and simple method to assess recalcitrance after pretreatment.

Lignocellulosic biomass is a complex network of polymers making up the cell walls of plants. It represents a feedstock of sustainable resources to be converted into fuels, chemicals, and materials. Because of its complex architecture, lignocellulose is a recalcitrant material that requires some pretreatments and several types of catalysts to be transformed efficiently. Gaining more knowledge in the architecture of plant cell walls is therefore important to understand and optimize transformation processes. For the first time, super-resolution imaging of poplar wood samples has been performed using the Stimulated Emission Depletion (STED) technique. In comparison to standard confocal images, STED reveals new details in cell wall structure, allowing the identification of secondary walls and middle lamella with fine details, while keeping open the possibility to perform topochemistry by the use of relevant fluorescent nano-probes. In particular, the deconvolution of STED images increases the signal-to-noise ratio so that images become very well defined. The obtained results show that the STED super-resolution technique can be easily implemented by using cheap commercial fluorescent rhodamine-PEG nano-probes which outline the architecture of plant cell walls due to their interaction with lignin. Moreover, the sample preparation only requires easily-prepared plant sections of a few tens of micrometers, in addition to an easily-implemented post-treatment of images. Overall, the STED super-resolution technique in combination with a variety of nano-probes can provide a new vision of plant cell wall imaging by filling in the gap between classical photon microscopy and electron microscopy., Competing Interests: The authors declare no conflict of interest.

Lignocellulosic biomass bioconversion is hampered by the structural and chemical complexity of the network created by cellulose, hemicellulose and lignin. Biological conversion of lignocellulose involves synergistic action of a large array of enzymes including the recently discovered lytic polysaccharide monooxygenases (LPMOs) that perform oxidative cleavage of cellulose. Using in situ imaging by synchrotron UV fluorescence, we have shown that the addition of AA9 LPMO (from Podospora anserina) to cellulases cocktail improves the progression of enzymes in delignified Miscanthus x giganteus as observed at tissular levels. In situ chemical monitoring of cell wall modifications performed by synchrotron infrared spectroscopy during enzymatic hydrolysis demonstrated that the boosting effect of the AA9 LPMO was dependent on the cellular type indicating contrasted recalcitrance levels in plant tissues. Our study provides a useful strategy for investigating enzyme dynamics and activity in plant cell wall to improve enzymatic cocktails aimed at expanding lignocelluloses biorefinery.

With the accumulation of scientific information in natural science, even experts can find difficult to keep integrating new piece of information. It is critical to explore modelling solutions able to capture information scattered in publications as a computable representation form. Traditional modelling techniques are important in that regard, but relying on numerical information comes with limitations for integrating results from distinct studies, high-level representations can be more suited. We present an approach to stepwise construct mechanistic explanation from selected scientific papers using the Qualitative Reasoning framework. As a proof of concept, we apply the approach to modelling papers about cellulose hydrolysis mechanism, focusing on the causal explanations for the decreasing of hydrolytic rate. Two explanatory QR models are built to capture classical explanations for the phenomenon. Our results show that none of them provides sufficient explanation for a set of basic experimental observations described in the literature. Combining the two explanations into a third one allowed to get a new and sufficient explanation for the experimental results. In domains where numerical data are scarce and strongly related to the experimental conditions, this approach can aid assessing the conceptual validity of an explanation and support integration of knowledge from different sources.

Lignocellulosic biomass is the only renewable carbon resource available in sufficient amount on Earth to go beyond the fossil-based carbon economy. Its transformation requires controlled breakdown of polymers into a set of molecules to make fuels, chemicals and materials. But biomass is a network of various inter-connected polymers which are very difficult to deconstruct optimally. In particular, saccharification potential of lignocellulosic biomass depends on several complex chemical and physical factors. For the first time, an easily measurable fluorescence properties of steam-exploded biomass samples from miscanthus, poplar and wheat straw was shown to be directly correlated to their saccharification potential. Fluorescence can thus be advantageously used as a predictive method of biomass saccharification. The loss in fluorescence occurring after the steam explosion pretreatment and increasing with pretreatment severity does not originate from the loss in lignin content, but rather from a decrease of the lignin β-aryl-ether linkage content. Fluorescence lifetime analysis demonstrates that monolignols making lignin become highly conjugated after steam explosion pretreatment. These results reveal that lignin chemical composition is a more important feature to consider than its content to understand and to predict biomass saccharification.

Background: Biorefining of lignocellulosic biomass has become one of the most valuable alternatives for the production of multi-products such as biofuels. Pretreatment is a prerequisite to increase the enzymatic conversion of the recalcitrant lignocellulose. However, there is still considerable debate regarding the key features of biomass impacting the cellulase accessibility. In this study, we evaluate the structural and chemical features of three different representative biomasses ( Miscanthus × giganteus , poplar and wheat straw), before and after steam explosion pretreatment at increasing severities, by monitoring chemical analysis, SEM, FTIR and 2D NMR., Results: Regardless the biomass type, combined steam explosion pretreatment with dilute sulfuric acid impregnation resulted in significant improvement of the cellulose conversion. Chemical analyses revealed that the pretreatment selectively degraded the hemicellulosic fraction and associated cross-linking ferulic acids. As a result, the pretreated residues contained mostly cellulosic glucose and lignin. In addition, the pretreatment directly affected the cellulose crystallinity but these variations were dependent upon the biomass type. Important chemical modifications also occurred in lignin since the β- O -4' aryl-ether linkages were found to be homolytically cleaved, followed by some recoupling/recondensation to β-β' and β-5' linkages, regardless the biomass type. Finally, 2D NMR analysis of the whole biomass showed that the pretreatment preferentially degraded the syringyl-type lignin fractions in miscanthus and wheat straw while it was not affected in the pretreated poplar samples., Conclusions: Our findings provide an enhanced understanding of parameters impacting biomass recalcitrance, which can be easily generalized to both woody and non-woody biomass species. Results indeed suggest that the hemicellulose removal accompanied by the significant reduction in the cross-linking phenolic acids and the redistribution of lignin are strongly correlated with the enzymatic saccharification, by loosening the cell wall structure thus allowing easier cellulase accessibility. By contrast, we have shown that the changes in the syringyl/guaiacyl ratio and the cellulose crystallinity do not seem to be relevant factors in assessing the enzymatic digestibility. Some biomass type-dependent and easily measurable FTIR factors are highly correlated to saccharification.

Background: The lignocellulosic cell wall network is resistant to enzymatic degradation due to the complex chemical and structural features. Pretreatments are thus commonly used to overcome natural recalcitrance of lignocellulose. Characterization of their impact on architecture requires combinatory approaches. However, the accessibility of the lignocellulosic cell walls still needs further insights to provide relevant information., Results: Poplar specimens were pretreated using different conditions. Chemical, spectral, microscopic and immunolabeling analysis revealed that poplar cell walls were more altered by sodium chlorite-acetic acid and hydrothermal pretreatments but weakly modified by soaking in aqueous ammonium. In order to evaluate the accessibility of the pretreated poplar samples, two fluorescent probes (rhodamine B-isothiocyanate-dextrans of 20 and 70 kDa) were selected, and their mobility was measured by using the fluorescence recovery after photobleaching (FRAP) technique in a full factorial experiment. The mobility of the probes was dependent on the pretreatment type, the cell wall localization (secondary cell wall and cell corner middle lamella) and the probe size. Overall, combinatory analysis of pretreated poplar samples showed that even the partial removal of hemicellulose contributed to facilitate the accessibility to the fluorescent probes. On the contrary, nearly complete removal of lignin was detrimental to accessibility due to the possible cellulose-hemicellulose collapse., Conclusions: Evaluation of plant cell wall accessibility through FRAP measurement brings further insights into the impact of physicochemical pretreatments on lignocellulosic samples in combination with chemical and histochemical analysis. This technique thus represents a relevant approach to better understand the effect of pretreatments on lignocellulose architecture, while considering different limitations as non-specific interactions and enzyme efficiency.

The interactions of proteins involved in plant cell wall hydrolysis, such as enzymes and CBMs, significantly determine their role and efficiency. In order to go beyond the characterization of interactions with simple ligands, bioinspired assemblies combined with the measurement of diffusion and interaction by FRAP offer a relevant alternative for highlighting the importance of different parameters related to the protein affinity and to the assembly.

Background: Enzymes degrading plant biomass polymers are widely used in biotechnological applications. Their efficiency can be limited by non-specific interactions occurring with some chemical motifs. In particular, the lignin component is known to bind enzymes irreversibly. In order to determine interactions of enzymes with their substrates, experiments are usually performed on isolated simple polymers which are not representative of plant cell wall complexity. But when using natural plant substrates, the role of individual chemical and structural features affecting enzyme-binding properties is also difficult to decipher., Results: We have designed and used lignified model assemblies of plant cell walls as templates to characterize binding properties of multi-modular cellulases. These three-dimensional assemblies are modulated in their composition using the three principal polymers found in secondary plant cell walls (cellulose, hemicellulose, and lignin). Binding properties of enzymes are obtained from the measurement of their mobility that depends on their interactions with the polymers and chemical motifs of the assemblies. The affinity of the multi-modular GH45 cellulase was characterized using a statistical analysis to determine the role played by each assembly polymer. Presence of hemicellulose had much less impact on affinity than cellulose and model lignin. Depending on the number of CBMs appended to the cellulase catalytic core, binding properties toward cellulose and lignin were highly contrasted., Conclusions: Model assemblies bring new insights into the molecular determinants that are responsible for interactions between enzymes and substrate without the need of complex analysis. Consequently, we believe that model bioinspired assemblies will provide relevant information for the design and optimization of enzyme cocktails in the context of biorefineries.

Lignocellulose-acting enzymes play a central role in the biorefinery of plant biomass to make fuels, chemicals and materials. These enzymes are often appended to carbohydrate binding modules (CBMs) that promote substrate targeting. When used in plant materials, which are complex assemblies of polymers, the binding properties of CBMs can be difficult to understand and predict, thus limiting the efficiency of enzymes. In order to gain more information on the binding properties of CBMs, some bioinspired model assemblies that contain some of the polymers and covalent interactions found in the plant cell walls have been designed. The mobility of three engineered CBMs has been investigated by FRAP in these assemblies, while varying the parameters related to the polymer concentration, the physical state of assemblies and the oligomerization state of CBMs. The features controlling the mobility of the CBMs in the assemblies have been quantified and hierarchized. We demonstrate that the parameters can have additional or opposite effects on mobility, depending on the CBM tested. We also find evidence of a relationship between the mobility of CBMs and their binding strength. Overall, bioinspired assemblies are able to reveal the unique features of affinity of CBMs. In particular, the results show that oligomerization of CBMs and the presence of ferulic acid motifs in the assemblies play an important role in the binding affinity of CBMs. Thus we propose that these features should be finely tuned when CBMs are used in plant cell walls to optimise bioprocesses.

Plant biomass is a potential resource of chemicals, new materials and biofuels that could reduce our dependency on fossil carbon, thus decreasing the greenhouse effect. However, due to its chemical and structural complexity, plant biomass is recalcitrant to green biological transformation by enzymes, preventing the establishment of integrated bio-refineries. In order to gain more knowledge in the architecture of plant cell wall to facilitate their deconstruction, many fluorescent probes bearing various fluorophores have been devised and used successfully to reveal the changes in structural motifs during plant biomass deconstruction, and the molecular interactions between enzymes and plant cell wall polymers. Fluorescent probes are thus relevant tools to explore plant cell wall deconstruction.

Progression of enzymes in lignocellulosic biomass is a crucial parameter in biorefinery processes, and it appears to be one of the limiting factors in optimizing lignocellulose degradation. In order to assay the importance of the chemical and structural features of the substrate matrix on enzyme mobility, we have designed bioinspired model assemblies of secondary plant cell walls, which have been used to measure the mobility of fluorescent probes while modifying different parameters (probe size, water content, polysaccharide concentration). The results were used to construct a model of probe mobility and to rank the parameters in order of importance. Water content and probe size were shown to have the greatest effect. Although these assemblies are simplified templates of the plant cell walls, our strategy paves the way for proposing new approaches for optimizing biomass saccharification, such as selecting enzymes with suitable properties.

Dynamics is a key feature of enzyme catalysis. Unfortunately, current experimental and computational techniques do not yet provide a comprehensive understanding and description of functional macromolecular motions. In this work, we have extended a novel computational technique, which combines molecular modeling methods and robotics algorithms, to investigate functional motions of protein loops. This new approach has been applied to study the functional importance of the so-called thumb-loop in the glycoside hydrolase family 11 xylanase from Thermobacillus xylanilyticus (Tx-xyl). The results obtained provide new insight into the role of the loop in the glycosylation/deglycosylation catalytic cycle, and underline the key importance of the nature of the residue located at the tip of the thumb-loop. The effect of mutations predicted in silico has been validated by in vitro site-directed mutagenesis experiments. Overall, we propose a comprehensive model of Tx-xyl catalysis in terms of substrate and product dynamics by identifying the action of the thumb-loop motion during catalysis.

For technical, environmental and economical reasons, industrial demands for process-fitted enzymes have evolved drastically in the last decade. Therefore, continuous efforts are made in order to get insights into enzyme structure/function relationships to create improved biocatalysts. Xylanases are hemicellulolytic enzymes, which are responsible for the degradation of the heteroxylans constituting the lignocellulosic plant cell wall. Due to their variety, xylanases have been classified in glycoside hydrolase families GH5, GH8, GH10, GH11, GH30 and GH43 in the CAZy database. In this review, we focus on GH11 family, which is one of the best characterized GH families with bacterial and fungal members considered as true xylanases compared to the other families because of their high substrate specificity. Based on an exhaustive analysis of the sequences and 3D structures available so far, in relation with biochemical properties, we assess biochemical aspects of GH11 xylanases: structure, catalytic machinery, focus on their "thumb" loop of major importance in catalytic efficiency and substrate selectivity, inhibition, stability to pH and temperature. GH11 xylanases have for a long time been used as biotechnological tools in various industrial applications and represent in addition promising candidates for future other uses., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Biomass from lignocellulose (LC) is a highly complex network of cellulose, hemicellulose, and lignin, which is considered to be a sustainable source of fuels, chemicals and materials. To achieve an environmental friendly and efficient LC upgrading, a better understanding of the LC architecture is necessary. We have devised some LC bioinspired model systems, based on arabinoxylan gels, in which mobility of dextrans and BSA grafted with FITC has been studied by FRAP. Our results indicate that the probes diffusion is more influenced by their hydrodynamic radius than by the gel mesh size. The addition of some cellulose nanocrystals (CNCs) decreases polymer chain mobility and has low effect on the probes diffusion, suggesting that the gels are better organized in the presence of CNCs, as shown by rheological measurements and scanning electronic microscopy observations. This demonstrates that the FRAP analysis can be a powerful tool to screen the architecture of LC model systems.

The crystal structure of the family GH-51 alpha- l-arabinofuranosidase from Thermobacillus xylanilyticus has been solved as a seleno-methionyl derivative. In addition, the structure of an inactive mutant Glu176Gln is presented in complex with a branched pentasaccharide, a fragment of its natural substrate xylan. The overall structure shows the two characteristic GH-51 domains: a catalytic domain that is folded into a (beta/alpha) 8-barrel and a C-terminal domain that displays jelly roll architecture. The pentasaccharide is bound in a groove on the surface of the enzyme, with the mono arabinosyl branch entering a tight pocket harboring the catalytic dyad. Detailed analyses of both structures and comparisons with the two previously determined structures from Geobacillus stearothermophilus and Clostridium thermocellum reveal important details unique to the Thermobacillus xylanilyticus enzyme. In the absence of substrate, the enzyme adopts an open conformation. In the substrate-bound form, the long loop connecting beta-strand 2 to alpha-helix 2 closes the active site and interacts with the substrate through residues His98 and Trp99. The results of kinetic and fluorescence titration studies using mutants underline the importance of this loop, and support the notion of an interaction between Trp99 and the bound substrate. We suggest that the changes in loop conformation are an integral part of the T. xylanilyticus alpha- l-arabinofuranosidase reaction mechanism, and ensure efficient binding and release of substrate.

GH-11 xylanases are highly specific and possess a thumb-shaped loop, a unique structure among enzymes with a jelly-roll scaffold. To investigate this structure, in vitro mutagenesis was performed on a GH-11 xylanase (Tx-Xyl) from Thermobacillus xylanilyticus. Targets were the conserved amino acids Pro(114)-Ser(115)-Ile(116) that are located at the thumb's tip and Thr(121) and Tyr(111), linker residues that connect the thumb to the main enzyme scaffold. Site-saturation mutagenesis provided an active variant that possesses a new triplet (Pro(114)-Gly(115)-Cys(116)), not found in naturally occurring GH-11 xylanases. The k(cat) value for xylan hydrolysis catalysed by this mutant was increased by 20%. Re-positioning of the thumb through the deletion of the linker residues produced different effects. As predicted by in silico analyses, deletion of Thr(121) had drastic consequences on activity, whereas deletion of Tyr(111) only affected (4-fold decrease) k(cat). Finally, deletion mutagenesis was used to create a thumbless variant that was almost catalytically inactive. Fluorescence titration with xylotetraose and xylopentaose revealed that this thumb-deleted xylanase retained the ability to bind substrates. This binding was comparable to that of the wild-type enzyme. Additionally, unlike wild-type Tx-Xyl, the thumb-deleted xylanase efficiently bound cellotetraose, although no cellulose hydrolysing activity was detected. Overall, these data show that the thumb is a key determinant for substrate selection and support previous data that suggest that it plays a role in the catalytic process.

Enzymatic hydrolysis constitutes an attractive strategy for biorefining of abundant, low-cost agricultural by-products such as wheat bran and straw. However, to adopt such an approach, efficient enzymes are required, in particular xylanases. To promote heat-induced disorganization of the complex cell wall network in wheat bran and thus increase enzymatic hydrolysis, we have attempted to improve the thermoresistance of a GH-11 xylanase that is already moderately thermostable. Using a previously described engineering strategy that involves the introduction of disulphide bridges, a mutant (Tx-xyl-SS3) displaying enhanced thermostability and thermoactivity was obtained. The half life at 70 degrees C (180 min) of Tx-xyl-SS3 is 10-fold greater than that of the wild type enzyme and its specific activity is almost doubled (3500 IU mg(-1)). Despite these improvements, Tx-xyl-SS3 was unsuitable for use at significantly higher reaction temperatures (i.e. 85-95 degrees C) and thus the initial objective of this study remained unaccomplished. However, unexpectedly even at the normal hydrolytic temperature (60 degrees C), Tx-xyl-SS3 was able to solubilize 50% of the wheat bran arabinoxylans, 10 points more than the wild type enzyme in parallel reactions. The data presented here show that this improvement is not directly linked to the increase in thermostability and/or thermoactivity, but rather to other unidentified changes to physico-chemical properties that may allow Tx-xyl-SS3 to better penetrate the cell wall network in wheat bran.

The external envelope of wheat grain (Triticum aestivum L. cv. Isengrain) is a natural composite whose tissular and cellular heterogeneity constitute a significant barrier for enzymatic cell wall disassembly. To better understand the way in which the cell wall network and tissular organization hamper enzyme penetration, we have devised a strategy based on in situ visualization of an active and an inactive form of a xylanase in whole-wheat bran and in three micro-dissected layers (the outer bran, the inner bran and the aleurone layer). The main aims of this study were to (1) evaluate the role of cuticular layers as obstacles to enzyme diffusion, (2) assess the impact of the cell wall network on xylanase penetration, (3) highlight wall heterogeneity. To conduct this study, we created by in vitro mutagenesis a hydrolytically inactive xylanase that displayed full substrate binding ability, as demonstrated by the calculation of dissociation constants (K(d)) using fluorescence titration. To examine enzyme penetration and action, immunocytochemical localization of the xylanases and of feebly substituted arabinoxylans (AXs) was performed following incubation of the bran layers, or whole bran with active and inactive isoforms of the enzyme for different time periods. The data obtained showed that the micro-dissected layers provided an increased accessible surface for the xylanase and that the enzyme-targeted cell walls were penetrated more quickly than those in intact bran. Examination of immunolabelling of xylanase indicated that the cuticle layers constitute a barrier for enzyme penetration in bran. Moreover, our data indicated that the cell wall network by itself physically restricts enzyme penetration. Inactive xylanase penetration was much lower than that of the active form, whose penetration was facilitated by the concomitant depletion of AXs in enzyme-sensitive cell walls.

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.

The role in activity of outer regions in the substrate binding cleft in alpha-amylases is illustrated by mutational analysis of Tyr(105) and Thr(212) localized at subsites -6 and +4 (substrate cleavage occurs between subsites -1 and +1) in barley alpha-amylase 1 (AMY1). Tyr(105) is conserved in plant alpha-amylases whereas Thr(212) varies in these and related enzymes. Compared with wild-type AMY1, the subsite -6 mutant Y105A has 140, 15, and <1% activity (k(cat)/K(m)) on starch, amylose DP17, and 2-chloro-4-nitrophenyl beta-d-maltoheptaoside, whereas T212Y at subsite +4 has 32, 370, and 90% activity, respectively. Thus engineering of aromatic stacking interactions at the ends of the 10-subsite long binding cleft affects activity very differently, dependent on the substrate. Y105A dominates in dual subsite -6/+4 [Y105A/T212(Y/W)]AMY1 mutants having almost retained and low activity on starch and oligosaccharides, respectively. Bond cleavage analysis of oligosaccharide degradation by wild-type and mutant AMY1 supports that Tyr(105) is critical for binding at subsite -6. Substrate binding is improved by T212(Y/W) introduced at subsite +4 and the [Y105A/T212(Y/W)]AMY1 double mutants synergistically enhanced productive binding of the substrate aglycone. The enzymatic properties of the series of AMY1 mutants suggest that longer substrates adopt several binding modes. This is in excellent agreement with computed distinct multiple docking solutions observed for maltododecaose at outer binding areas of AMY1 beyond subsites -3 and +3.