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Abstract Background The CBM13 family comprises carbohydrate-binding modules that occur mainly in enzymes and in several ricin-B lectins. The ricin-B lectin domain resembles the CBM13 module to a large extent. Historically, ricin-B lectins and CBM13 proteins were considered completely distinct, despite their structural and functional similarities. Results In this data mining study, we investigate structural and functional similarities of these intertwined protein groups. Because of the high structural and functional similarities, and differences in nomenclature usage in several databases, confusion can arise. First, we demonstrate how public protein databases use different nomenclature systems to describe CBM13 modules and putative ricin-B lectin domains. We suggest the introduction of a novel CBM13 domain identifier, as well as the extension of CAZy cross-references in UniProt to guard the distinction between CAZy and non-CAZy entries in public databases. Since similar problems may occur with other lectin families and CBM families, we suggest the introduction of novel CBM InterPro domain identifiers to all existing CBM families. Second, we investigated phylogenetic, nomenclatural and structural similarities between putative ricin-B lectin domains and CBM13 modules, making use of sequence similarity networks. We concluded that the ricin-B/CBM13 superfamily may be larger than initially thought and that several putative ricin-B lectin domains may display CAZyme functionalities, although biochemical proof remains to be delivered. Conclusions Ricin-B lectin domains and CBM13 modules are associated groups of proteins whose database semantics are currently biased towards ricin-B lectins. Revision of the CAZy cross-reference in UniProt and introduction of a dedicated CBM13 domain identifier in InterPro may resolve this issue. In addition, our analyses show that several proteins with putative ricin-B lectin domains show very strong structural similarity to CBM13 modules. Therefore ricin-B lectin domains and CBM13 modules could be considered distant members of a larger ricin-B/CBM13 superfamily.

Background: The CBM13 family comprises carbohydrate-binding modules that occur mainly in enzymes and in several ricin-B lectins. The ricin-B lectin domain resembles the CBM13 module to a large extent. Historically, ricin-B lectins and CBM13 proteins were considered completely distinct, despite their structural and functional similarities. Results: In this data mining study, we investigate structural and functional similarities of these intertwined protein groups. Because of the high structural and functional similarities, and differences in nomenclature usage in several databases, confusion can arise. First, we demonstrate how public protein databases use different nomenclature systems to describe CBM13 modules and putative ricin-B lectin domains. We suggest the introduction of a novel CBM13 domain identifier, as well as the extension of CAZy cross-references in UniProt to guard the distinction between CAZy and non-CAZy entries in public databases. Since similar problems may occur with other lectin families and CBM families, we suggest the introduction of novel CBM InterPro domain identifiers to all existing CBM families. Second, we investigated phylogenetic, nomenclatural and structural similarities between putative ricin-B lectin domains and CBM13 modules, making use of sequence similarity networks. We concluded that the ricin-B/CBM13 superfamily may be larger than initially thought and that several putative ricin-B lectin domains may display CAZyme functionalities, although biochemical proof remains to be delivered. Conclusions: Ricin-B lectin domains and CBM13 modules are associated groups of proteins whose database semantics are currently biased towards ricin-B lectins. Revision of the CAZy cross-reference in UniProt and introduction of a dedicated CBM13 domain identifier in InterPro may resolve this issue. In addition, our analyses show that several proteins with putative ricin-B lectin domains show very strong structural similarity to CBM13 modules. Therefore ricin-B lectin domains and CBM13 modules could be considered distant members of a larger ricin-B/CBM13 superfamily. [ABSTRACT FROM AUTHOR]

[Objective]This study is aimed to explore the binding ability of different sources of CBM to beech-xylanan, and to fuse exogenous CBM with high binding ability to the C-terminal and N-terminal of Streptomyces L10904 xylanase(XYN), to explore the effects of exogenous CBM on the enzymatic properties of xylanase.[Method]First, through the substrate adsorption method, the concentration of CBM in the solution before and after adsorption was detected by Coomassie Brilliant blue G250 method, and the substrate binding rate of CBM was calculated. CBM1 and CBM4 with better xylan binding ability were screened. In order to explore the effect of the fusion location of CBM with high substrate binding ability on the enzymatic properties of xylanase, CBM1 and CBM4 were fused with the C-terminal and N-terminal of XYN by flexible binding peptide, and four recombinant enzymes were obtained by expression in Escherichia coli BL21(DE3). They were named CBM1-XYN, XYN-CBM1, CBM4-XYN, and XYN-CBM4.[Result]The binding rates of CBM1 and CBM4 to xylan were 89% and 95%, respectively. The specific activities of XYN, CBM1-XYN, XYN-CBM1, CBM4-XYN and XYN-CBM4 were 32 274.81, 49 342.21, 602.48, 230.42 and 2 362.24 U/mg, respectively, measured at 60℃ and pH 7.0. The specific activities of CBM1-XYN were 1.5 times higher than the specific activities of XYN. The analysis of enzymatic properties showed that CBM1 improved the temperature stability and pH stability of XYN, XYN and CBM1-XYN were incubated at 60℃ for 1 h, and the residual enzyme activity of CBM1-XYN and XYN was 81% and 28%, respectively. In the pH range of 3-11, CBM1-XYN maintained more than 90% enzyme activity after incubation at 4℃ for 12 h.[Conclusion]Streptomyces derived xylanase was heterogeneically expressed in E. coli BL21(DE3), and CBM1 and CBM4 with high substrate binding rates were screened. CBM was successfully fused to XYN by protein fusion technology, and CBM1-XYN with improved enzymatic properties was obtained, which improved the temperature stability, pH tolerance and specific enzyme activity of xylanase. [ABSTRACT FROM AUTHOR]

To date, due to the low accessibility of enzymes to xanthan substrates, the enzymolysis of xanthan remains deficient, which hinders the industrial production of functional oligoxanthan. To enhance the enzymatic affinity against xanthan, the essential role of two carbohydrate binding modules—MiCBMx and PspCBM84, respectively, derived from Microbacterium sp. XT11 and Paenibacillus sp. 62047—in catalytic properties of endotype xanthanase MiXen were investigated for the first time. Basic characterizations and kinetic parameters of different recombinants revealed that, compared with MiCBMx, PspCBM84 dramatically increased the thermostability of endotype xanthanase, and endowed the enzyme with higher substrate affinity and catalytic efficiency. Notably, the activity of endotype xanthanase was increased by 16 times after being fused with PspCBM84. In addition, the presence of both CBMs obviously enabled endotype xanthanase to produce more oligoxanthan, and xanthan digests prepared by MiXen-CBM84 showed better antioxidant activity due to the higher content of active oligosaccharides. The results of this work lay a foundation for the rational design of endotype xanthanase and the industrial production of oligoxanthan in the future. [ABSTRACT FROM AUTHOR]

The inefficient decomposition of polysaccharides, particularly branched polysaccharides limits their large-scale industrial applications. Further understanding and modification of glycoside hydrolases (GHs) processivity is expected to overcome this limitation. Here, a novel xanthan-binding CBM (Mi XBM), which was supposed to alter the processivity of GHs, was systematically characterized. Phylogeny and structure analyses indicated that Mi XBM is closely related to putative polysaccharide side chain-binding modules. Quantitative binding assays further revealed that Mi XBM probably has a high affinity for xanthan side chain via a variable loop site. Moreover, catalytic performance demonstrated that xanthanase chimeras containing Mi XBM promote highly efficient hydrolysis of xanthan because of improved substrate accessibility. Notably, Mi XBM was observed to enhance the processivity of xanthanase, owing to its high substrate affinity to the repeating unit xanthan. Furthermore, sequential hydrolysis of xanthan by xanthanases with varying processivity resulted in significantly increased hydrolytic efficiency and focused oligoxanthans array. These results expand understanding of CBM-substrate recognition and shed light on efficient degradation of other regularly branched polysaccharides using modified GHs. [Display omitted] [ABSTRACT FROM AUTHOR]

Lignocellulosic biomass, rich in cellulose, hemicellulose, and lignin, offers a sustainable source for biofuels and and production of other materials such as polymers, paper, fabrics, bioplastics and biofertilizers. However, its complex structure hinders efficient conversion. Chemical, enzymatic, and microbial methods aim to unlock the trapped sugars and phenols. The rumen microbiome, a fascinating ecosystem within ruminant animals, holds particular promise. The Hungate 1000 project sequenced 410 microbial genomes from the rumen, enabling in silico screening for lignocellulolytic enzymes. This approach saves time and resources, supporting the development of sustainable bioconversion technologies aligned with the UN's 2030 agenda goals. Analysis of these 410 predicted proteomes revealed diverse carbohydrate-active enzymes (CAZymes) and carbohydrate-binding modules (CBMs) across various microorganisms. Notably, Firmicutes and Bacteroidetes dominated CAZyme and CBM production, suggesting collaborative efforts among different phyla during degradation. The presence of CBM50 and chitinases hints at the ability to utilize chitin from fungal cell walls. Interestingly, the absence of ligninolytic auxiliary activity enzymes reaffirms the rumen microbiome's incapability of directly degrading lignin. However, enzymes facilitating the loosening of the cell wall by cleaving lignin-hemicellulose linkages were identified. This suggests a strategy for making cellulose more accessible to hydrolytic enzymes. This study highlights the intricate relationship between rumen microbes, contributing necessary enzymes for plant cell wall deconstruction in this unique environment. Additionally, it underlines the power of in silico techniques for analyzing big data, paving the way for advancements in sustainable bioconversion., Competing Interests: Declarations. Competing interests: On behalf of all authors, the corresponding author states that there is no conflict of interest., (© 2025. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

Bioproduction of diverse N -acetyl chitooligosaccharides from chitin is of great value. In the study, a novel GH family 18 bifunctional chitinase gene (PsChi82) from Paenibacillus shirakamiensis was identified, expressed and biochemically characterized. PsChi82 was most active at pH 5.0, and 55 °C, and displayed remarkable pH stability with the broad pH range of 3.0–12.0. It showed high chitosanase activity of 10.6 U mg−1 and diverse hydrolysis products of GlcNAc, (GlcNAc) 2 , GlcN-GlcNAc and (GlcN) 2 -GlcNAc, which may facilitate comprehensively understanding of structure-function relationships of N -acetyl COSs. Three engineered variants were then expressed and characterized. Among them, PsChi82-CBM26 possessed specific activity of 25.1 U mg−1 against colloidal chitin, which was 2.1 folds higher than that of PsChi82. The diverse N -acetyl COSs were subsequently produced by PsChi82-CBM26 with a sugar content of 23.2 g L−1. These excellent properties may make PsChi82-CBM26 potentially useful for N -acetyl COSs production in the food and chemical industries. [Display omitted] • A novel bifunctional chitinase gene (PsChi82) was successfully expressed in E. coli. • The great pH stability and high chitosanase activity of PsChi82 were determined. • PsChi82 showed diverse hydrolysis products including two types of paCOSs. • Three engineered variants were designed, and PsChi82-CBM26 possessed high activity. • An efficient bioprocess for diverse N -acetyl COSs production was developed. [ABSTRACT FROM AUTHOR]

The emergence of antibiotic-resistant bacteria is a critical worldwide healthcare problem. In the specific case of wound care, new and effective alternatives to currently available solutions are urgently needed. Cellulose-based dressings, for example, could be made more attractive if rendered antimicrobial. This work proposes a new strategy to modify cellulose-based materials with the short antimicrobial hexapeptide MP196 (RWRWRW - NH 2) that relies on a biomolecular recognition approach based on carbohydrate binding modules (CBMs). Specifically, we focused on the modification of hydrogels, paper, and microfibrillated cellulose (MFC) with fusions of the CBM3 from Clostridium thermocellum (C. thermocellum) with derivatives of MP196. The fusions are prepared by promoting the formation of a disulfide bond between Cys-terminated derivatives of MP196 and a CBM3 that is pre-anchored in the materials. The CBM3-MP196-modified materials displayed antibacterial activity against Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) that was significantly higher when compared with the activity of materials prepared by physical adsorption of MP196. The biomolecular strategy provides a more favorable orientation, exposure, and distancing of the peptide from the matrix. This versatile concept provides a toolbox for the functionalization of cellulose materials of different origins and architectures with a broad choice in peptides. Functionalization under mild biological conditions avoids further purification steps, allowing for translational research and multiple applications as drug delivery systems, scaffolds for tissue engineering and biomaterials. The emergence of antibiotic-resistant bacteria is a critical worldwide healthcare problem. In the specific case of wound care, new and effective alternatives to currently available solutions are urgently needed. This work proposes a new strategy to modify cellulose-based materials with a short antimicrobial hexapeptide that relies on a biomolecular recognition approach based on carbohydrate binding modules. The modified materials displayed antibacterial activity against both Gram-negative and Gram-positive bacteria. The biomolecular strategy provides a favorable orientation, exposure, and distancing of the peptide from the matrix. This versatile concept offers a toolbox for the functionalization of different cellulose materials with a broad choice in peptides. Functionalization under mild biological conditions avoids further purification steps, allowing for translational research and multiple applications. [Display omitted] [ABSTRACT FROM AUTHOR]

Glycoside hydrolase family 5 subfamily 8 (GH5_8) mannanases belong to Firmicutes, Actinomycetia, and Proteobacteria. The presence or absence of carbohydrate-binding modules (CBMs) present a striking difference. While various GH5_8 mannanases need a CBM for binding galactomannans, removal of the CBM did not affect activity of some, whereas it in other cases reduced the catalytic efficiency due to increased KM. Here, monomodular GH5_8 mannanases from Eubacterium siraeum (EsGH5_8) and Xanthomonas citri pv. aurantifolii (XcGH5_8) were produced and characterized to clarify if GH5_8 mannanases from Firmicutes and Proteobacteria without CBM(s) possess distinct properties. EsGH5_8 showed a remarkably high temperature optimum of 55 °C, while XcGH5_8 had an optimum at 30 °C. Both enzymes were highly active on carob galactomannan and konjac glucomannan. Notably, EsGH5_8 was equally active on both substrates, whereas XcGH5_8 preferred galactomannan. The KM values were comparable with those of catalytic domains of truncated GH5_8s, while the turn-over numbers (kcat) were in the higher end. Notably, XcGH5_8 bound to but did not degrade insoluble ivory nut mannan. The findings support the hypothesis that GH5_8 mannanases with CBMs target insoluble mannans found in plant cell walls and seeds, while monomodular GH5_8 members have soluble mannans and mannooligosaccharides as primary substrates. [ABSTRACT FROM AUTHOR]

Perennial grasses, such as Panicum maximum, are important alternatives to dedicated energy crops for bioethanol production. This study investigates whether future climate conditions could influence P. maximum cell‐wall structure and hydrolytic performance. To analyze interactions with environmental factors in field conditions, a combined Free‐air Temperature and CO2 Controlled Enhancement (Trop‐T‐FACE) facility was used to investigate the isolated and combined effect of elevated atmospheric CO2 concentration (eC) (600 μmol.mol−1) and elevated temperature (eT) by 2 °C more than the ambient temperature, on cell‐wall composition, cellulose crystallinity, accessibility, and hydrolysis yields. The elevated temperature treatments (eT and eT + eC) exhibited the most pronounced effects on the P. maximum cell wall. Warming reduced the starch content and the crystallinity index (CI) of cellulose and increased the cellulose content. Fluorescent protein‐tagged carbohydrate‐binding modules analysis demonstrated that warming improved total cellulose surface exposure/accessibility in eT and eT + eC by 181% and 132%, respectively. Consequently, glucan conversion yields were improved by 7.07% and 5.37%, showing that warming led to lower recalcitrance in P. maximum biomass, which positively affects its use in biorefineries. This work therefore provides important information from an ecological and economic point of view, allowing us to understand the mitigation process applied by this forage grass under future climate conditions. It might assist in selecting tropical forage grasses that are efficiently adapted to climate change, with a positive effect on bioenergy production. © 2021 Society of Chemical Industry and John Wiley & Sons, Ltd [ABSTRACT FROM AUTHOR]

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.

[Display omitted] • Fermentation methods partially determined transcriptional response of strains. • Trichoderma asperellum T-1 effectively deconstructed the structure of wheat straw. • Secretion of carbohydrate-binding modules enhanced straw deconstruction by T-1. • T. asperellum T-1 is suitable for both solid state and submerged fermentation. • T. reesei QM6a is preferred used in solid state fermentation. Microorganisms harvest energy from agricultural waste by degrading its structure. By comparing with Trichoderma reesei QM6a in cellulase production, straw deconstruction and transcriptome response, Trichoderma asperellum T-1 was identified to be prioritized for the fermentation of natural straw. Cellulase activity of T-1 was 50%–102% higher than QM6a. And the degradation rate of hemicellulose and ligin in wheat straw by T-1 reached 40% and 42%. Time-driven changes in the gene expression of extracellular proteins involved in polysaccharide, xylan, and hemicellulose metabolism and hydrolysis indicated that T-1 positively responded in both solid state fermentation and submerged fermentation for lignocellulose degradation. A significantly enriched category encoding carbohydrate-binding modules is considered critical for the deconstruction of the natural structure by T-1. The findings highlight the superiority of T. asperellum T-1 in straw fermentation, base on which, the construction of efficient microbial agents is expected to enhance the utilization of biomass. [ABSTRACT FROM AUTHOR]

Plant cell wall-derived biomass serves as a renewable source of energy and materials with increasing importance. The cell walls are biomacromolecular assemblies defined by a fine arrangement of different classes of polysaccharides, proteoglycans, and aromatic polymers and are one of the most complex structures in Nature. One of the most challenging tasks of cell biology and biomass biotechnology research is to image the structure and organization of this complex matrix, as well as to visualize the compartmentalized, multiplayer biosynthetic machineries that build the elaborate cell wall architecture. Better knowledge of the plant cells, cell walls, and whole tissue is essential for bioengineering efforts and for designing efficient strategies of industrial deconstruction of the cell wall-derived biomass and its saccharification. Cell wall-directed molecular probes and analysis by light microscopy, which is capable of imaging with a high level of specificity, little sample processing, and often in real time, are important tools to understand cell wall assemblies. This review provides a comprehensive overview about the possibilities for fluorescence label-based imaging techniques and a variety of probing methods, discussing both well-established and emerging tools. Examples of applications of these tools are provided. We also list and discuss the advantages and limitations of the methods. Specifically, we elaborate on what are the most important considerations when applying a particular technique for plants, the potential for future development, and how the plant cell wall field might be inspired by advances in the biomedical and general cell biology fields. [ABSTRACT FROM AUTHOR]

Chemoenzymatic approaches using carbohydrate‐active enzymes (CAZymes) offer a promising avenue for the synthesis of glycans like oligosaccharides. Here, we report a novel chemoenzymatic route for cellodextrins synthesis employed by chimeric CAZymes, akin to native glycosyltransferases, involving the unprecedented participation of a "non‐catalytic" lectin‐like domain or carbohydrate‐binding modules (CBMs) in the catalytic step for glycosidic bond synthesis using β‐cellobiosyl donor sugars as activated substrates. CBMs are often thought to play a passive substrate targeting role in enzymatic glycosylation reactions mostly via overcoming substrate diffusion limitations for tethered catalytic domains (CDs) but are not known to participate directly in any nucleophilic substitution mechanisms that impact the actual glycosyl transfer step. This study provides evidence for the direct participation of CBMs in the catalytic reaction step for β‐glucan glycosidic bonds synthesis enhancing activity for CBM‐based CAZyme chimeras by >140‐fold over CDs alone. Dynamic intradomain interactions that facilitate this poorly understood reaction mechanism were further revealed by small‐angle X‐ray scattering structural analysis along with detailed mutagenesis studies to shed light on our current limited understanding of similar transglycosylation‐type reaction mechanisms. In summary, our study provides a novel strategy for engineering similar CBM‐based CAZyme chimeras for the synthesis of bespoke oligosaccharides using simple activated sugar monomers. [ABSTRACT FROM AUTHOR]

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 enzymemediated 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights • Bioextrusion improved enzymatic hydrolysis efficiency of raw corn crop residues. • FTCM assisted to study the surface cellulose accessibility profile. • Negative impact of high S/L ratio was not due to the lack of exposed cellulose. • Alkaline pretreatment strongly limits bioextrusion's positive influence. Abstract 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. [ABSTRACT FROM AUTHOR]

Abstract The enzymatic modification of polysaccharides has attracted much research attention, as it raises the possibility of using highly selective reactions performed by naturally designed proteins for practical purposes. However, achieving high performance of enzymes for the degradation of insoluble natural polysaccharides with highly ordered structures, such as cellulose and chitin, remains challenging. In the case of chitopolysaccharides, their chemical structure is amenable to hydrolysis by many hydrolases specific to different glucans or even by proteases or lipases. Some of these enzymes are even more active than chitinases and chitosanases in the hydrolysis of soluble chitosan, thereby opening up many opportunities for wide-scale and cost-effective enzymolysis of chitopolysaccharides. When compared with enzymatic treatment data for chitosan, data for chitin is limited, largely because of the poor accessibility of this polymer to enzymes. Most studies on enzymatic hydrolysis of polysaccharides have focused on cellulose, which is a recalcitrant substrate, and have confirmed the importance of cellulose-binding modules in facilitating enzyme action against this insoluble substrate. This review summarizes recent data concerning the mechanism of action of specific and nonspecific enzymes in the hydrolysis of chitin and chitosan and regarding the application of a large number of different enzymes in chitooligosaccharide production, as well as information about the disruption of the polysaccharide crystalline structure by accessory proteins. Possible reasons for the extraordinary enzymatic digestibility of chitin and chitosan are discussed. Highlights • Enzymatic hydrolysis of chitin as a recalcitrant insoluble substrate is discussed. • Mechanism of action of specific and nonspecific enzymes on chitopolysaccharides is analyzed. • Recent data on accessory proteins is summarized. [ABSTRACT FROM AUTHOR]

The microbial decomposition and utilization of lignocellulosic biomass present in the plant tissues are driven by a series of carbohydrate active enzymes (CAZymes) acting in concert. As the non-catalytic domains widely found in the modular CAZymes, carbohydrate-binding modules (CBMs) are intimately associated with catalytic domains (CDs) that effect the diverse hydrolytic reactions. The CBMs function as auxiliary components for the recognition, adhesion, and depolymerization of the complex substrate mediated by the associated CDs. Therefore, CBMs are deemed as significant biotools available for enzyme engineering, especially to facilitate the enzymatic hydrolysis of dense and insoluble plant tissues to acquire more fermentable sugars. This review aims at presenting the taxonomies and biological properties of the CBMs currently curated in the CAZy database. The molecular mechanisms that CBMs use in assisting the enzymatic hydrolysis of plant polysaccharides and the regulatory factors of CBM-substrate interactions are outlined in detail. In addition, guidelines for the rational designs of CBM-fused CAZymes are proposed. Furthermore, the potential to harness CBMs for industrial applications, especially in enzymatic pretreatment of the recalcitrant lignocellulose, is evaluated. It is envisaged that the ideas outlined herein will aid in the engineering and production of novel CBM-fused enzymes to facilitate efficient degradation of lignocellulosic biomass to easily fermentable sugars for production of value-added products, including biofuels. • Microbial CBMs are categorized based on substrate-binding pockets. • Mechanisms of three CBMs in the hydrolysis of plant biomass are illustrated. • Feasible approaches to construct effective CBM-fused enzymes are proposed. • Strategies to exploit CBMs for the hydrolysis of lignocellulose are summarized. [ABSTRACT FROM AUTHOR]

FmEG from Fomitiporia mediterranea is a non-modular endoglucanase composed of a 24-amino acids extension and 13-amino acids linker-like peptide at the N-terminus and a 312-amino acids GH5 catalytic domain (CD) at the C-terminus. In this study, six FmEG derivatives with deletion of N-terminal fragments or fusion with an extra family 1 carbohydrate-binding module (CBM1) was constructed in order to evaluate the contribution of CBM1 to FmEG processivity and catalytic activity. FmEG showed a weak processivity and released cellobiose (G2) and cellotriose (G3) as main end products, and cellotriose (G4) as minor end product from filter paper (FP), but more amount of G4 was released from regenerated amorphous cellulose (RAC). All derivatives had similar activity on carboxymethylcellulose (CMC) with the same optimal pH (7.0) and temperature (50 °C). However, fusing an extra CBM1 to FmEG△24 or FmEG△37 with flexible peptide significantly improved its processivity and catalytic activity to FP and RAC. Overall, 1.79- and 1.84-fold increases in the soluble/insoluble product ratio on FP, and 1.38- and 1.39-fold increases on RAC, compared to FmEG△24, were recorded for CBM1-FmEG△24 and CBM1-linker-FmEG△24, respectively. Meanwhile, they displayed 2.64- and 2.67-fold more activity on RAC, and 1.68- and 1.77-fold on FP, respectively. Similar improvement was also obtained for CBM1-linker-FmEG△37 as compared with FmEG△37. Interestingly, fusion of an extra CBM1 with FmEG also caused an alteration of cleavage pattern on insoluble celluloses. Our results suggest that such improvements in processivity and catalytic activity may arise from CBM1 binding affinity. The N-terminal 24- or 37-amino acids may serve as linker for sufficient spatial separation of the two domains required for processivity and catalytic activity. In addition, deletion of the N-terminal 24- or 37-amino acids led to significant reduction in thermostability but not the enzymatic activity. [ABSTRACT FROM AUTHOR]

The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted β-glucans, β-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize β-glucans and β-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose. [ABSTRACT FROM AUTHOR]

Family 1 carbohydrate-binding modules (CBMs) are ubiquitous components of multimodular fungal enzymes that degrade plant cell wall polysaccharides and bind specifically to cellulose. Native glycosylation of family 1 CBMs has been shown to substantially impact multiple physical properties, including thermal and proteolytic stability and cellulose binding affinity. To gain molecular insights into the changes in CBM properties upon glycosylation, solution structures of two glycoforms of a Trichoderma reesei family 1 CBM were studied by NMR spectroscopy: a glycosylated family 1 CBM with a mannose group attached to both Thr1 and Ser3 and a second family 1 CBM with single mannose groups attached to Thr1, Ser3 and Ser14. The structures clearly reveal that monosaccharides at both Ser3 and Ser14 on family 1 CBMs present additional cellulose binding platforms, similar to well-characterized aromatic residues at the binding interface, which align to the cellulose surface. These results are in agreement with previous experimental work demonstrating that glycans at Ser3 and Ser14 impart significant improvements in binding affinity. Additionally, detailed analysis of the NMR structures and molecular simulations indicates that the protein backbone of the CBM is not significantly altered by attachment of monosaccharides, and that the mannose attached to Ser14 may be more flexible than the mannose at Ser3. Overall, the present study reveals how family 1 CBM structures are affected by covalent attachment of monosaccharides, which are likely important post-translational modifications of these common subdomains of fungal plant cell wall degrading enzymes. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are ubiquitous in nature and at the core of many physiological processes of profound importance to health and disease. Specificity in protein-carbohydrate interactions is conferred by carbohydrate-binding modules ( CBMs) that can accurately discriminate among the multitude of saccharides found in nature, thus targeting proteins to their particular substrates. Family 14 carbohydrate-binding modules ( CBM14s), more specifically, are short modules that bind explicitly to chitin, the second most abundant carbohydrate in nature. Although considerable effort has been placed in elucidating protein-carbohydrate interactions at the molecular level for biological and biotechnological applications, in contrast the evolutionary relationships among these modules are minimally understood. Using the CBM14 family as an example, here we describe one of the first global molecular evolutionary analyses of a CBM family across all domains of life, with an emphasis on its origin, taxonomic distribution and pattern of diversification as a result of gene and module duplication, and positive selection. Our genome-wide searches recovered an impressive number of CBM14s from diverse lineages across nearly all domains of life. However, their highly disseminated distribution in taxa outside the Opisthokonta group strongly suggests a later evolutionary origin and elevated rates of inter- and intra-domain horizontal gene transfer. Moreover, accelerated rates of asymmetric gains and losses reveal a dynamic mode of birth-and-death evolution, whereas positive selection acting on paralogous CBM14-containing proteins suggest changes in substrate specificity and an increase in the functional promiscuity of this ancient CBM family. The importance of these results is discussed. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Protein-carbohydrate interactions are ubiquitous in nature and at the core of many physiological processes of profound importance to health and disease. Specificity in protein-carbohydrate interactions is conferred by carbohydrate- binding modules (CBMs) that can accurately discriminate among the multitude of saccharides found in nature, thus targeting proteins to their particular substrates. Family 14 carbohydrate-binding modules (CBM14s), more specifically, are short modules that bind explicitly to chitin, the second most abundant carbohydrate in nature. Although considerable effort has been placed in elucidating protein-carbohydrate interactions at the molecular level for biological and biotechnological applications, in contrast the evolutionary relationships among these modules are minimally understood. Using the CBM14 family as an example, here we describe one of the first global molecular evolutionary analyses of a CBM family across all domains of life, with an emphasis on its origin, taxonomic distribution and pattern of diversification as a result of gene and module duplication, and positive selection. Our genome-wide searches recovered an impressive number of CBM14s from diverse lineages across nearly all domains of life. However, their highly disseminated distribution in taxa outside the Opisthokonta group strongly suggests a later evolutionary origin and elevated rates of inter- and intra-domain horizontal gene transfer. Moreover, accelerated rates of asymmetric gains and losses reveal a dynamic mode of birth-and-death evolution, whereas positive selection acting on paralogous CBM14-containing proteins suggest changes in substrate specificity and an increase in the functional promiscuity of this ancient CBM family. The importance of these results is discussed. [ABSTRACT FROM AUTHOR]

Carbohydrate-binding modules (CBMs) are the noncatalytic modules that assist functions of the catalytic modules in carbohydrate-active enzymes, and they are usually discrete structural domains in larger multimodular enzymes. CBMs often occur in tandem in different alginate lyases belonging to the CBM families 13, 16, and 32. However, none of the currently known CBMs in alginate lyases specifically bind to an internal alginate chain. In our investigation of the multidomain alginate lyase Dp0100 carrying several ancillary domains, we identified an alginate-binding domain denoted TM6-N4 using protein truncation analysis. The structure of this CBM domain was determined at 1.35 Å resolution. TM6-N4 exhibited an overall β-sandwich fold architecture with two antiparallel β-sheets. We identified an extended binding groove in the CBM using site-directed mutagenesis, docking, and surface electrostatic potential analysis. Affinity analysis revealed that residues of Lys10, Lys22, Lys25, Lys27, Lys31, Arg36, and Tyr159 located on the bottom or the wall of the shallow groove are responsible for alginate binding, and isothermal titration calorimetry analyses indicated that the binding cleft consists of six subsites for sugar recognition. This substrate binding pattern is typical for type B CBM, and it represents the first CBM domain that specifically binds internal alginate chain. Phylogenetic analysis supports that TM6-N4 constitutes the founding member of a new CBM family denoted as CBM96. Our reported structure not only facilitates the investigation of the CBM-alginate ligand recognition mechanism but also inspires the utilization of the CBM domain in biotechnical applications., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Ruminococcus albus, a cellulolytic bacterium, is a critical member of the rumen community. Ruminococcus albus lacks a classical cellulosome complex, but it possesses a unique family 37 carbohydrate-binding module (CBM37), which is integrated into a variety of carbohydrate-active enzymes. We developed a potential molecular tool for functional phylotyping of the R. albus population in the rumen, based on a variable region in the cel48A gene. cel48A encodes a single copy of the CBM37-associated family 48 glycoside hydrolase in all known strains of this bacterium. A segment of the cel48A gene was amplified from rumen metagenomic samples of four bovines, and its abundance and diversity were evaluated. Analysis of the obtained sequences revealed the co-existence of multiple functional phylotypes of cel48A in all four animals. These included sequences identical or similar to those of R. albus isolates (reference strains), as well as several novel sequences. The dominant cel48A type varied among animals. This method can be used for detection of intraspecific diversity of R. albus in metagenomic samples. Together with scaC, a previously reported gene marker for R. flavefaciens, we present a set of two species--specific markers for phylotyping of Ruminococci in the herbivore rumen. [ABSTRACT FROM AUTHOR]

In this review, the present knowledge on the occurrence of cellulases, with a special emphasis on the presence of carbohydrate-binding modules (CBMs) in various fungal strains, has been summarized. The importance of efficient fungal cellulases is growing due to their potential uses in biorefinery processes where lignocellulosic biomasses are converted to platform sugars and further to biofuels and chemicals. Most secreted cellulases studied in detail have a bimodular structure containing an active core domain attached to a CBM. CBMs are traditionally been considered as essential parts in cellulases, especially in cellobiohydrolases. However, presently available genome data indicate that many cellulases lack the binding domains in cellulose-degrading organisms. Recent data also demonstrate that CBMs are not necessary for the action of cellulases and they solely increase the concentration of enzymes on the substrate surfaces. On the other hand, in practical industrial processes where high substrate concentrations with low amounts of water are employed, the enzymes have been shown to act equally efficiently with and without CBM. Furthermore, available kinetic data show that enzymes without CBMs can desorb more readily from the often lignaceous substrates, that is, they are not stuck on the substrates and are thus available for new actions. In this review, the available data on the natural habitats of different wood-degrading organisms (with emphasis on the amount of water present during wood degradation) and occurrence of cellulose-binding domains in their genome have been assessed in order to identify evolutionary advantages for the development of CBM-less cellulases in nature. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: N- or C-terminal fusions of red-fluorescent protein (RFP) with various fungal cellulose-binding domains (CBDs) belonging to carbohydrate binding module (CBM) family 1 were expressed in a Pichia pastoris expression system, and the resulting fusion proteins were used to examine the feasibility of large-scale affinity purification of CBD-tagged proteins on cellulose columns. We found that RFP fused with CBD from Trichoderma reesei CBHI (CBD Tr CBHI) was expressed at up to 1.2g/l in the culture filtrate, which could be directly injected into the cellulose column. The fusion protein was tightly adsorbed on the cellulose column in the presence of a sufficient amount of ammonium sulfate and was efficiently eluted with pure water. Bovine serum albumin (BSA) was not captured under these conditions, whereas both BSA and the fusion protein were adsorbed on a phenyl column, indicating that the cellulose column can be used for the purification of not only hydrophilic proteins but also for hydrophobic proteins. Recovery of various fusion proteins exceeded 80%. Our results indicate that protein purification by expression of a target protein as a fusion with a fungal family 1 CBD tag in a yeast expression system, followed by affinity purification on a cellulose column, is simple, effective and easily scalable. [Copyright &y& Elsevier]

Here, a 2.0 Å resolution X-ray structure of Clostridium thermocellum cellulase K family 4 carbohydrate-binding module (CelK CBM4) is reported. The resulting structure was refined to an R factor of 0.212 and an Rfree of 0.274. Structural analysis shows that this new structure is very similar to the previously solved structure of C. thermocellum CbhA CBM4. Most importantly, these data support the previously proposed notion of an extended binding pocket using a novel tryptophan-containing loop that may be highly conserved in clostridial CBM4 proteins. [ABSTRACT FROM AUTHOR]

Abstract: In general, plant cell wall degrading enzymes are modular proteins containing catalytic domains linked to one or more non-catalytic carbohydrate-binding modules (CBMs). Xyn10B from Clostridium thermocellum is a typical modular enzyme containing an N-terminal family 22 CBM (CBM22-1), a family 10 glycoside hydrolase catalytic domain (GH10), a second CBM22 (CBM22-2), a dockerin sequence and a C-terminal family 1 carbohydrate esterase (CE1) catalytic domain. The structure of the N-terminal bi-modular CBM22-1–GH10 component of Xyn10B has been determined using a SeMet derivative by SAD to 2.5Å. The data was extended to 2.0Å for the non-SeMet mutant complexed with xylohexaose. CBM22-1–GH10 is a 60kDa protein with an E337A mutation to render the GH10 subunit inactive. Three of the six xylose residues of xylohexaose are shown to be bound in the inactivated GH10 substrate binding cleft, with the other three sugars presumably disordered in the solvent channel. The protein is a dimer in the asymmetric unit with extensive surface contacts between the two GH10 modules and between the CBM22-1 and GH10 modules. Residues from helix H4 of the GH10 module provide the major contacts by fitting into the minor groove of the CBM22-1 module. The orientation of CBM22-1 is such that it would allow the substrate to be loosely bound and subsequently delivered to the active site in a processive manner. [ABSTRACT FROM AUTHOR]

We report a new method for the direct labeling and visualization of crystalline cellulose using quantum dots (QDs) directed by carbohydrate-binding modules (CBMs). Two type-I (surface binding) CBMs belonging to families 2 and 3a were cloned and expressed with dual histidine tags at the N- and C-termini. Semiconductor (CdSe)ZnS QDs were used to label these CBMs following their binding to Valonia cellulose crystals. Using this approach, we demonstrated that QDs are linearly arrayed on cellulose, which implies that these CBMs specifically bind to a planar face of cellulose. Direct imaging has further shown that different sizes (colors) of QDs can be used to label CBMs bound to cellulose. Furthermore, the binding density of QDs arrayed on cellulose was modified predictably by selecting from various combinations of CBMs and QDs of known dimensions. This approach should be useful for labeling and imaging cellulose-containing materials precisely at the molecular scale, thereby supporting studies of the molecular mechanisms of lignocellulose conversion for biofuels production. [ABSTRACT FROM AUTHOR]

The crystal structure of the uncharacterized protein SO2946 from Shewanella oneidensis MR-1 was determined with single-wavelength anomalous diffraction (SAD) and refined to 2.0 Å resolution. The SO2946 protein consists of a short helical N-terminal domain and a large C-terminal domain with the “jelly-roll” topology. The protein assembles into a propeller consisting of three C-terminal blades arranged around a central core formed by the N-terminal domains. The function of SO2946 could not be inferred from the sequence since the protein represents an orphan with no sequence homologs, but the protein’s structure bears a fold similar to that of proteins containing carbohydrate-binding modules. Features such as fold conservation, the presence of a conserved groove and a metal binding region are indicative that SO2946 may be an enzyme and could be involved in binding carbohydrate molecules. [ABSTRACT FROM AUTHOR]

This article focuses on the molecular determinants that drive the binding and recognition of polysaccharides to the carbohydrate-binding modules (CBMs) from family 11. The CBMs are noncatalytic modules of a high-hierarchy multisubunit complex called cellulosome, which are known to be crucial for the efficient degradation of polysaccharides. Their function and behavior has not yet been fully clarified, and taking into account the bioeconomical interest in this subject it has become a challenging research problem. Our computational results point to the fact that the binding interface of the CBM11 can bind only one single polysaccharide chain, which is in agreement with other CBMs from type B. The central binding site has affinity for polysaccharides with more than four subunits, and there are four main residues that have a central role in this interaction: Asp99, Arg126, Asp128, and Asp146. Furthermore, there are three tyrosine residues, Tyr22, Tyr53, and Tyr129 that are crucial for the guiding and packing of the polysaccharide to the charged regions. In this article, we have also compared the structure of the CBM11 with other CBMs families from type B and found many similarities, which suggest similar binding activities and functions. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2008 [ABSTRACT FROM AUTHOR]

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 CBM2 b-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 CBM2 b-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 CBM2 b-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 CBM2 b-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 CBM2 b-1-2 transformants was less severe. Altered cell expansion was also observed in most parts of the CBM29-1-2 stem whereas for the CBM2 b-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. [ABSTRACT FROM AUTHOR]

In the present study, the carbohydrate-binding module (CBM) coding sequences of the cellulosomes of 13 thermophilic, cellulolytic, anaerobic, ethanol-producing bacterial isolates having some variations in their growth and cellulose degradation capacities were amplified by PCR and then sequenced. The sequence analysis of the amplicons revealed that CBMs of 7 of the isolates including the isolate 7-9-1 with the highest capacity of cellulose degradation in solid medium have 100% identity in both nucleotide and amino acid sequences to CipB of Clostridium thermocellum in the compared regions. On the other hand, CBM of the isolate 7-1-2, also having a high cellulolytic activity, was found to differ for as much as 66 amino acid residues out of 100 (66%). The isolate 7-9-4 with a relatively low cellulose-degrading capacity also displayed amino acid variation for this protein, but only for 4 out of 118 residues. [ABSTRACT FROM AUTHOR]

Glycoside hydrolases often possess carbohydrate-binding modules (CBMs) in addition to their catalytic domains, which help target the enzymes to appropriate substrates and thereby increase their catalytic efficiency. Sialidases hydrolyse the release of sialic acid from a variety of glycoconjugates and play significant roles in the pathogenesis of a number of important diseases. The sialidase from Micromonospora viridifaciens has a CBM which recognizes galactose. The CBM is linked to the catalytic domain by an immunoglobulin-like domain, resulting in the galactose binding site sitting above the catalytic site, suggesting an interplay between the two sites. By studying nine crystallographically independent structures of the M. viridifaciens sialidase, the relative flexibility of the three domains was analysed. A detailed study is also presented of the recognition of galactose and lactose by the M. viridifaciens CBM. The striking structure of this sialidase suggests a role for the CBM in binding to galactose residues unmasked by the adjacent catalytic site. [ABSTRACT FROM AUTHOR]

The structural and thermodynamic basis for carbohydrate–protein recognition is of considerable importance. NCP-1, which is a component of the Piromyces equi cellulase/hemicellulase complex, presents a provocative model for analyzing how structural and mutational changes can influence the ligand specificity of carbohydrate-binding proteins. NCP-1 contains two “family 29” carbohydrate-binding modules designated CBM29-1 and CBM29-2, respectively, that display unusually broad specificity; the proteins interact weakly with xylan, exhibit moderate affinity for cellulose and mannan, and bind tightly to the β-1,4-linked glucose-mannose heteropolymer glucomannan. The crystal structure of CBM29-2 in complex with cellohexaose and mannohexaose identified key residues involved in ligand recognition. By exploiting this structural information and the broad specificity of CBM29-2, we have used this protein as a template to explore the evolutionary mechanisms that can lead to significant changes in ligand specificity. Here, we report the properties of the E78R mutant of CBM29-2, which displays ligand specificity that is different from that of wild-type CBM29-2; the protein retains significant affinity for cellulose but does not bind to mannan or glucomannan. Significantly, E78R exhibits a stoichiometry of 0.5 when binding to cellohexaose, and both calorimetry and ultracentrifugation show that the mutant protein displays ligand-mediated dimerization in solution. The three-dimensional structure of E78R in complex with cellohexaose reveals the intriguing molecular basis for this “dimeric” binding mode that involves the lamination of the oligosaccharide between two CBM molecules. The 2-fold screw axis of the ligand is mirrored in the orientation of the two protein domains with adjacent sugar rings stacking against the equivalent aromatic residues in the binding site of each protein molecule of the molecular sandwich. The sandwiching of an oligosaccharide chain between two protein modules, leading to ligand-induced formation of the binding site, represents a completely novel mechanism for protein–carbohydrate recognition that may mimic that displayed by naturally dimeric protein–carbohydrate interactions. [Copyright &y& Elsevier]