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A pre-hydrolysis Kraft pulping (PHK) process that was used to make dissolving pulp was enhanced by replacing conventional white liquor (WL) neutralization with green liquor (GL) neutralization prior to Kraft pulping. This resulted in a 10% increase in dissolving pulp production, and significant chemical savings, without compromising pulp reactivity. When the possible influence of the alkaline charge on fibre properties was assessed using methods such as viscosity, Simon's stain, Size Exclusion Chromatography (SEC) and SEM, it was apparent that stronger alkaline treatments (WL) resulted increased cellulose degradation, a lower cellulose DP and a slightly larger surface area. When these methods were complemented with an assay based on the selective binding of site-specific carbohydrate-binding modules (CBMs), it was apparent that green liquor (GL) neutralization resulted in an increase in less-ordered cellulose being exposed. This likely contributed to its higher reactivity despite its lower overall surface area.

Significance It is recognized that an optimized mixture of endo- and exo-glucanases, beta-glucosidases, and accessory enzymes need to work cooperatively to result in effective cellulose hydrolysis. However, a key factor that impacts hydrolysis is the effectiveness of enzyme access to the cellulose. A quantitative method based on fluorescence-tagged CBMs and CLSM was developed to assess the cellulose structure, quantify its accessibility, and elucidate how this might influence the efficiency of cellulose hydrolysis. This work provides insights into the substrate–enzyme interactions that occur during enzyme-mediated hydrolysis of cellulose., Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

International audience; Lignocellulosic biomass is a sustainable source of renewable substrate to produce low carbon footprint energy and materials. Biomass conversion is usually performed in two steps: a biomass pretreatment for improving cellulose accessibility followed by enzymatic hydrolysis of cellulose. In this study we investigated the efficiency of a bioextrusion pretreatment (extrusion in the presence of cellulase enzyme) for production of reducing sugars from corn crop agricultural residues. Our results demonstrate that bioextrusion increased the reducing sugar conversion yield by at least 94% at high solid/liquid ratio (14%-40%). Monitoring biomass surface with carbohydrate-binding modules (FTCM-depletion assay) revealed that well known negative impact of high solid/liquid ratio on conversion yield is not due to the lack of exposed cellulose which was abundant under such conditions. Bioextrusion was found to be less efficient on alkaline pretreated biomass but being a mild and solvent limiting pretreatment, it might help to minimize the waste stream.

Poly(ethylene terephthalate) (PET) is one of the most widely applied synthetic polymers, but its hydrophobicity is challenging for many industrial applications. Biotechnological modification of PET surface can be achieved by PET hydrolyzing cutinases. In order to increase the adsorption towards their unnatural substrate, the enzymes are fused to carbohydrate-binding modules (CBMs) leading to enhanced activity. In this study, we identified novel PET binding CBMs and characterized the CBM-PET interplay. We developed a semi-quantitative method to detect CBMs bound to PET films. Screening of eight CBMs from diverse families for PET binding revealed one CBM that possesses a high affinity towards PET. Molecular dynamics (MD) simulations of the CBM–PET interface revealed tryptophan residues forming an aromatic triad on the peptide surface. Their interaction with phenyl rings of PET is stabilized by additional hydrogen bonds formed between amino acids close to the aromatic triad. Furthermore, the ratio of hydrophobic to polar contacts at the interface was identified as an important feature determining the strength of PET binding of CBMs. The interaction of CBM tryptophan residues with PET was confirmed experimentally by tryptophan quenching measurements after addition of PET nanoparticles to CBM. Our findings are useful for engineering PET hydrolyzing enzymes and may also find applications in functionalization of PET. Electronic supplementary material The online version of this article (10.1007/s00253-019-09760-9) contains supplementary material, which is available to authorized users.

We have recently presented a sequential treatment method, in which steam explosion (STEX) was followed by hydrotropic extraction (HEX), to selectively fractionate cellulose, hemicellulose, and lignin in hardwood into separate process streams. However, above a treatment severity threshold, the structural alterations in the cellulose-enriched fraction appeared to restrict the enzymatic hydrolyzability and delignification efficiency. To better understand the ultrastructural changes in the cellulose, hardwood chips were treated by single (STEX or HEX) and combined treatments (STEX and HEX), and the cellulose accessibility quantified with carbohydrate-binding modules (CBMs) that bind preferentially to crystalline (CBM2a) and paracrystalline cellulose (CBM17). Fluorescent-tagged versions of the CBMs were used to map the spatial distribution of cellulose substructures with confocal laser scanning microscopy. With increasing severities, STEX increased the apparent crystallinity (CBM2a/CBM17-ratio) and overall accessibility (CBM2aH6 + CBM17) of the cellulose, whereas HEX demonstrated the opposite trend. The respective effects could also be discerned in the combined treatments where increasing severities further resulted in higher hemicellulose dissolution and, although initially beneficial, in stagnating accessibility and hydrolyzability. This study suggests that balancing the severities in the two treatments is required to maximize the fractionation and simultaneously achieve a reactive and accessible cellulose that is readily hydrolyzable., Elucidation of cellulose structural properties that restrict enzymatic hydrolysis in sustainable fractionation processes for hardwoods.

Genome sequencing has revealed substantial variation in the predicted abilities of individual species within animal gut microbiota to metabolize the complex carbohydrates comprising dietary fiber. At the same time, a currently limited body of functional studies precludes a richer understanding of how dietary glycan structures affect the gut microbiota composition and community dynamics. Here, using biochemical and biophysical techniques, we identified and characterized differences among recombinant proteins from syntenic xyloglucan utilization loci (XyGUL) of three Bacteroides and one Dysgonomonas species from the human gut, which drive substrate specificity and access to distinct polysaccharide side chains. Enzymology of four syntenic glycoside hydrolase family 5 subfamily 4 (GH5_4) endo-xyloglucanases revealed surprising differences in xyloglucan (XyG) backbone cleavage specificity, including the ability of some homologs to hydrolyze congested branched positions. Further, differences in the complement of GH43 alpha-l-arabinofuranosidases and GH95 alpha-l-fucosidases among syntenic XyGUL confer distinct abilities to fully saccharify plant species-specific arabinogalactoxyloglucan and/or fucogalactoxyloglucan. Finally, characterization of highly sequence-divergent cell surface glycan-binding proteins (SGBPs) across syntenic XyGUL revealed a novel group of XyG oligosaccharide-specific SGBPs encoded within select Bacteroides. IMPORTANCE The catabolism of complex carbohydrates that otherwise escape the endogenous digestive enzymes of humans and other animals drives the composition and function of the gut microbiota. Thus, detailed molecular characterization of dietary glycan utilization systems is essential both to understand the ecology of these complex communities and to manipulate their compositions, e.g., to benefit human health. Our research reveals new insight into how ubiquitous members of the human gut microbiota have evolved a set of microheterogeneous gene clusters to efficiently respond to the structural variations of plant xyloglucans. The data here will enable refined functional prediction of xyloglucan utilization among diverse environmental taxa in animal guts and beyond.

Carbohydrate-binding modules (CBMs) are small components of several enzymes, which present an independent fold and function, and specific carbohydrate-binding activity. Their major function is to bind the enzyme to the substrate enhancing its catalytic activity, especially in the case of insoluble substrates. The immense diversity of CBMs, together with their unique properties, has long raised their attention for many biotechnological applications. Recombinant DNA technology has been used for cloning and characterizing new CBMs. In addition, it has been employed to improve the purity and availability of many CBMs, but mainly, to construct bi-functional CBM-fused proteins for specific applications. This review presents a comprehensive summary of the uses of CBMs recombinantly produced from heterologous organisms, or by the original host, along with the latest advances. Emphasis is given particularly to the applications of recombinant CBM-fusions in: (a) modification of fibers, (b) production, purification and immobilization of recombinant proteins, (c) functionalization of biomaterials and (d) development of microarrays and probes., Fundação para a Ciência e a Tecnologia (FCT), Portugal (grants SFRH/BDP/63831/ 2009 and SFRH/BPD/73850/2010, respectively). The authors thank the FCT GlycoCBMs Project REF. PTDC/AGR-FOR/3090/2012 — FCOMP-01- 0124-FEDER-027948, the FCT Strategic Project PEst-OE/EQB/LA0023/ 2013, and the Project “BioInd — Biotechnology and Bioengineering for improved Industrial and Agro-Food processes”, REF. NORTE-07-0124- FEDER-000028 Co-funded by the Programa Operacional Regional do Norte (ON.2 — O Novo Norte), QREN, FEDER.

International audience; For more than a century, filamentous fungi have been used for the production of a wide variety of endogenous enzymes of industrial interest. More recently, with the use of genetic engineering tools developed for these organisms, this use has expanded for the production of nonnative heterologous proteins. In this review, an overview is given of examples describing the production of a special class of these proteins, namely chimeric proteins. The production of two types of chimeric proteins have been explored: (a) proteins grafted for a specific substrate-binding domain and (b) fusion proteins containing two separate enzymatic activities. Various application areas for the use of these chimeric proteins are described.

Polysaccharide utilization loci (PUL) within the genomes of resident human gut Bacteroidetes are central to the metabolism of the otherwise indigestible complex carbohydrates known as “dietary fiber.” However, functional characterization of PUL lags significantly behind sequencing efforts, which limits physiological understanding of the human-bacterial symbiosis. In particular, the molecular basis of complex polysaccharide recognition, an essential prerequisite to hydrolysis by cell surface glycosidases and subsequent metabolism, is generally poorly understood. Here, we present the biochemical, structural, and reverse genetic characterization of two unique cell surface glycan-binding proteins (SGBPs) encoded by a xyloglucan utilization locus (XyGUL) from Bacteroides ovatus, which are integral to growth on this key dietary vegetable polysaccharide. Biochemical analysis reveals that these outer membrane-anchored proteins are in fact exquisitely specific for the highly branched xyloglucan (XyG) polysaccharide. The crystal structure of SGBP-A, a SusD homolog, with a bound XyG tetradecasaccharide reveals an extended carbohydrate-binding platform that primarily relies on recognition of the β-glucan backbone. The unique, tetra-modular structure of SGBP-B is comprised of tandem Ig-like folds, with XyG binding mediated at the distal C-terminal domain. Despite displaying similar affinities for XyG, reverse-genetic analysis reveals that SGBP-B is only required for the efficient capture of smaller oligosaccharides, whereas the presence of SGBP-A is more critical than its carbohydrate-binding ability for growth on XyG. Together, these data demonstrate that SGBP-A and SGBP-B play complementary, specialized roles in carbohydrate capture by B. ovatus and elaborate a model of how vegetable xyloglucans are accessed by the Bacteroidetes., IMPORTANCE The Bacteroidetes are dominant bacteria in the human gut that are responsible for the digestion of the complex polysaccharides that constitute “dietary fiber.” Although this symbiotic relationship has been appreciated for decades, little is currently known about how Bacteroidetes seek out and bind plant cell wall polysaccharides as a necessary first step in their metabolism. Here, we provide the first biochemical, crystallographic, and genetic insight into how two surface glycan-binding proteins from the complex Bacteroides ovatus xyloglucan utilization locus (XyGUL) enable recognition and uptake of this ubiquitous vegetable polysaccharide. Our combined analysis illuminates new fundamental aspects of complex polysaccharide recognition, cleavage, and import at the Bacteroidetes cell surface that may facilitate the development of prebiotics to target this phylum of gut bacteria.

Background Enzymes still comprise a major part of ethanol production costs from lignocellulose raw materials. Irreversible binding of enzymes to the residual substrate prevents their reuse and no efficient methods for recycling of enzymes have so far been presented. Cellulases without a carbohydrate-binding module (CBM) have been found to act efficiently at high substrate consistencies and to remain non-bound after the hydrolysis. Results High hydrolysis yields could be obtained with thermostable enzymes of Thermoascus aurantiacus containing only two main cellulases: cellobiohydrolase I (CBH I), Cel7A and endoglucanase II (EG II), Cel5A. The yields were decreased by only about 10% when using these cellulases without CBM. A major part of enzymes lacking CBM was non-bound during the most active stage of hydrolysis and in spite of this, produced high sugar yields. Complementation of the two cellulases lacking CBM with CBH II (CtCel6A) improved the hydrolysis. Cellulases without CBM were more sensitive during exposure to high ethanol concentration than the enzymes containing CBM. Enzymes lacking CBM could be efficiently reused leading to a sugar yield of 90% of that with fresh enzymes. The applicability of cellulases without CBM was confirmed under industrial ethanol production conditions at high (25% dry matter (DM)) consistency. Conclusions The results clearly show that cellulases without CBM can be successfully used in the hydrolysis of lignocellulose at high consistency, and that this approach could provide new means for better recyclability of enzymes. This paper provides new insight into the efficient action of CBM-lacking cellulases. The relationship of binding and action of cellulases without CBM at high DM consistency should, however, be studied in more detail.

Introduction Cellulases are of great interest for application in biomass degradation, yet the molecular details of the mode of action of glycoside hydrolases during degradation of insoluble cellulose remain elusive. To further improve these enzymes for application at industrial conditions, it is critical to gain a better understanding of not only the details of the degradation process, but also the function of accessory modules. Method We fused a carbohydrate-binding module (CBM) from family 2a to two thermophilic endoglucanases. We then applied neutron reflectometry to determine the mechanism of the resulting enhancements. Results Catalytic activity of the chimeric enzymes was enhanced up to three fold on insoluble cellulose substrates as compared to wild type. Importantly, we demonstrate that the wild type enzymes affect primarily the surface properties of an amorphous cellulose film, while the chimeras containing a CBM alter the bulk properties of the amorphous film. Conclusion Our findings suggest that the CBM improves the efficiency of these cellulases by enabling digestion within the bulk of the film.

International audience; Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

We have compared heterologous expression of two types of carbohydrate binding module (CBM) in tobacco cell walls. These are the promiscuous CBM29 modules (a tandem CBM29-1-2 and its single derivative CBM29-2), derived from a non-catalytic protein1, NCP1, of the Piromyces equi cellulase/hemicellulase complex, and the less promiscuous tandem CBM2b-1-2 from the Cellulomonas fimi xylanase 11A. CBM-labelling studies revealed that CBM29-1-2 binds indiscriminately to every tissue of the wild-type tobacco stem whereas binding of CBM2b-1-2 was restricted to vascular tissue. The promiscuous CBM29-1-2 had much more pronounced effects on transgenic tobacco plants than the less promiscuous CBM2b-1-2. Reduced stem elongation and prolonged juvenility, resulting in delayed flower development, were observed in transformants expressing CBM29-1-2 whereas such growth phenotypes were not observed for CBM2b-1-2 plants. Histological examination and electron microscopy revealed layers of collapsed cortical cells in the stems of CBM29-1-2 plants whereas cellular deformation in the stem cortical cells of CBM2b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2b-1-2 stem this was observed in the xylem cells only. The cellulose content of the transgenic plants was not altered. These results support the hypothesis that CBMs can modify cell wall structure leading to modulation of wall loosening and plant growth.

Resistance against the leaf mold fungus Cladosporium fulvum is mediated by the tomato Cf proteins which belong to the class of receptor-like proteins and indirectly recognize extracellular avirulence proteins (Avrs) of the fungus. Apart from triggering disease resistance, Avrs are believed to play a role in pathogenicity or virulence of C. fulvum. Here, we report on the avirulence protein Avr4, which is a chitin-binding lectin containing an invertebrate chitin-binding domain (CBM14). This domain is found in many eukaryotes, but has not yet been described in fungal or plant genomes. We found that interaction of Avr4 with chitin is specific, because it does not interact with other cell wall polysaccharides. Avr4 binds to chitin oligomers with a minimal length of three N-acetyl glucosamine residues. In vitro, Avr4 protects chitin against hydrolysis by plant chitinases. Avr4 also binds to chitin in cell walls of the fungi Trichoderma viride and Fusarium solani f. sp. phaseoli and protects these fungi against normally deleterious concentrations of plant chitinases. In situ fluorescence studies showed that Avr4 also binds to cell walls of C. fulvum during infection of tomato, where it most likely protects the fungus against tomato chitinases, suggesting that Avr4 is a counter-defensive virulence factor.