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Abstract The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application.

Abstract Laccases are multi-copper oxidases that are usually composed of three Cu-oxidase domains. Domains one and three house the copper binding sites, and the second domain is involved in forming a substrate-binding cleft. However, Streptomyces species are found to have small laccases (SLAC) that lack one of the three Cu-oxidase domains. This type of SLAC with interesting lignocellulose bioconversion activities has not been reported in Aspergillus niger. In our research, we explored the expression and engineering of the SLAC from Streptomyces leeuwenhoekii C34 in A. niger. Genes encoding two versions of the SLAC were expressed. One encoding the SLAC in its native form and a second encoding the SLAC fused to two N-terminal CBM1 domains. The latter is a configuration also known for specific yeast laccases. Both SLAC variants were functionally expressed in A. niger as shown by in vitro activity assays and proteome analysis. Laccase activity was also analyzed toward bioconversion of lignocellulosic rice straw. From this analysis it was clear that the SLAC activity improved the efficiency of saccharification of lignocellulosic biomass by cellulase enzyme cocktails.

Abstract Cellulosic materials are attracting increasing research interest because of their abundance, biocompatibility, and biodegradability, making them suitable in multiple industrial and medical applications. Functionalization of cellulose is usually required to improve or expand its properties to meet the requirements of different applications. Cellulose-binding domains (CBDs) found in various proteins have been shown to be powerful tools in the functionalization of cellulose materials. In this review, we firstly introduce the structural characteristics of commonly used CBDs belonging to carbohydrate-binding module families 1, 2 and 3. Then, we summarize four main kinds of methodologies for employing CBDs to modify cellulosic materials (i.e., CBD only, genetic fusion, non-covalent linkage and covalent linkage). Via different approaches, CBDs have been used to improve the material properties of cellulose, immobilize enzymes for biocatalysis, and design various detection tools. To achieve industrial applications, researches for lowering the production cost of CBDs, improving their performance (e.g., stability), and expanding their application scenarios are still in need. Graphical abstract

The glycoside hydrolase family 3 (GH3) β-glucosidases from filamentous fungi are crucial industrial enzymes facilitating the complete degradation of lignocellulose, by converting cello-oligosaccharides and cellobiose into glucose. Understanding the diverse domain organization is essential for elucidating their biological roles and potential biotechnological applications. This research delves into the variability of domain organization within GH3 β-glucosidases. Two distinct configurations were identified in fungal GH3 β-glucosidases, one comprising solely the GH3 catalytic domain, and another incorporating the GH3 domain with a C-terminal fibronectin type III (Fn3) domain. Notably, Streptomyces filamentous bacteria showcased a separate clade of GH3 proteins linking the GH3 domain to a carbohydrate binding module from family 2 (CBM2). As a first step to be able to explore the role of accessory domains in β-glucosidase activity, a screening system utilizing the well-characterised Aspergillus niger β-glucosidase gene (bglA) in bglA deletion mutant host was developed. Based on this screening system, reintroducing the native GH3-Fn3 gene successfully expressed the gene allowing detection of the protein using different enzymatic assays. Further investigation into the role of the accessory domains in GH3 family proteins, including those from Streptomyces, will be required to design improved chimeric β-glucosidases enzymes for industrial application. [ABSTRACT FROM AUTHOR]

Understanding the relation between enzyme domain structure and catalytic activity is crucial for optimal engineering of novel enzymes for lignocellulose bioconversion. Xylanases with varying specificities are commonly used to valorise the hemicellulose arabinoxylan (AX), yet characterization of specific arabinoxylanases remain limited. Two homologous GH5_34 arabinoxylanases, Hh Xyn5A and Ct Xyn5A, in which the two domains are connected by a 40-residue linker, exhibit distinct activity on AX, yielding different reaction product patterns, despite high sequence identity, conserved active sites and similar domain composition. In this study, the carbohydrate binding module 6 (CBM6), or the inter domain linker together with CBM6, were swapped to investigate their influence on hydrolytic activity and oligosaccharide product pattern on cereal AXs. The variants, with only CBM6 swapped, displayed reduced activity on commercial wheat and rye AX, as well as on extracted oat fibre, compared to the original enzymes. Additionally, exchange of both linker and CBM6 resulted in a reduced ratio of enzyme produced in soluble form in Escherichia coli cultivations, causing loss of activity of both Hh Xyn5A and Ct Xyn5A variants. Analysis of oligosaccharide product patterns applying HPAEC–PAD revealed a decreased number of reaction products for Ct Xyn5A with swapped CBM6, which resembled the product pattern of Hh Xyn5A. These findings emphasize the importance of the CBM6 interactions with the linker and the catalytic domain for enzyme activity and specificity, and underlines the role of the linker in enzyme structure organisation and product formation, where alterations in linker interactions with the catalytic and/or CBM6 domains, influence enzyme-substrate association and specificity. [ABSTRACT FROM AUTHOR]

Cellulosic materials are attracting increasing research interest because of their abundance, biocompatibility, and biodegradability, making them suitable in multiple industrial and medical applications. Functionalization of cellulose is usually required to improve or expand its properties to meet the requirements of different applications. Cellulose-binding domains (CBDs) found in various proteins have been shown to be powerful tools in the functionalization of cellulose materials. In this review, we firstly introduce the structural characteristics of commonly used CBDs belonging to carbohydrate-binding module families 1, 2 and 3. Then, we summarize four main kinds of methodologies for employing CBDs to modify cellulosic materials (i.e., CBD only, genetic fusion, non-covalent linkage and covalent linkage). Via different approaches, CBDs have been used to improve the material properties of cellulose, immobilize enzymes for biocatalysis, and design various detection tools. To achieve industrial applications, researches for lowering the production cost of CBDs, improving their performance (e.g., stability), and expanding their application scenarios are still in need. [ABSTRACT FROM AUTHOR]

The red fluorescent protein mCherry is widely used as a tagged protein in intracellular protein positioning and dynamic tracing due to its stable characteristics in color and monomer molecule. In this study, mCherry was used as a tag to explore the cell wall-directed binding of the carbohydrate-binding module (CBM), in order to intuitively demonstrate that the fusion protein anchored on the cell wall. Two constitutive expression vectors harboring CBM-mCherry fusion gene were constructed for rice transformation. The results of fluorescent signal detection showed that both the ubiquitin promoter and the CaMV35S promoter could drive the expression of mCherry fusion gene in seeds, leaves, and roots. The results of subcellular localization by different methods, such as cytological observation, immunofluorescence detection and protoplast observation, displayed that the fluorescence signals were concentrated on the cell walls of the root, stem, leaf, and vascular bundle sheath in rice. It indicated that the CBM could mediate the red fluorescent protein anchoring on the cell wall, and the cell wall may be a good subcellular structure for accumulation of exogenous proteins in future. Key message: The fusion gene SP-CBM11-mCherry was transformed into rice, and the subcellular localization of fusion protein indicated the red fluorescent protein mCherry anchored on the cell wall. [ABSTRACT FROM AUTHOR]

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Carbohydrate active enzymes are valuable tools in cereal processing to valorize underutilized side streams. By solubilizing hemicellulose and modifying the fiber structure, novel food products with increased nutritional value can be created. In this study, a novel GH5_34 subfamily arabinoxylanase from Herbinix hemicellulosilytica , Hh Xyn5A, was identified, produced and extensively characterized, for the intended exploitation in cereal processing to solubilize potential prebiotic fibers: arabinoxylo-oligosaccharides. The purified two-domain Hh Xyn5A (catalytic domain and CBM6) demonstrated high storage stability, showed a melting temperature T m of 61°C and optimum reaction conditions were determined to 55°C and pH 6.5 on wheat arabinoxylan. Hh Xyn5A demonstrated activity on various commercial cereal arabinoxylans and produced prebiotic AXOS, whereas the sole catalytic domain of Hh Xyn5A did not demonstrate detectable activity. Hh Xyn5A demonstrated no side activity on oat β-glucan. In contrast to the commercially available homolog Ct Xyn5A, Hh Xyn5A gave a more specific HPAEC–PAD oligosaccharide product profile when using wheat arabinoxylan and alkali extracted oat bran fibers as the substrate. Results from multiple sequence alignment of GH5_34 enzymes, homology modeling of Hh Xyn5A and docking simulations with ligands XXXA3, XXXA3XX and X5 concluded that the active site of Hh Xyl5A catalytic domain is highly conserved and can accommodate both shorter and longer ligands. However, significant structural dissimilarities between Hh Xyn5A and Ct Xyn5A in the binding cleft of CBM6, due to the lack of important ligand-interacting residues, is suggested to cause the observed differences in substrate specificity and product formation. [ABSTRACT FROM AUTHOR]

Starch, a versatile ingredient used in various industries, often requires modification for improvements in its physicochemical and functional properties for utilization. Although carbohydrate-binding modules (CBMs) are non-catalytic domains that recognize and bind to the substrate without any enzymatic activity, CBMs are commonly found in the starch-modifying enzyme, pullulanase (PulA). Herein, we identified two PulAs from Deinococcus radiodurans and Deinococcus wulumuqiensis that differed in activity only by CBM composition. Structural comparison analysis showed unique substrate-binding coordination of CBM b1. The analysis with a deletion mutant (ΔCBM b1 -DrPulA), and a chimeric mutant (CBM b1 -DwPulA) revealed that CBM b1 can improve amylopectin but not pullulan hydrolysis. Moreover, CBM b1 introduction increased the hydrolysis activity of raw waxy corn starch by 60% which leads to the formation of distinct α-glucan microparticles (GMP). GMP produced using CBM b1 -DwPulA exhibited B-type crystallinity, similar to that produced by DwPulA. However, a relatively high proportion of short-length maltooligosaccharides was observed in the GMP produced by CBM b1 -DwPulA compared with that produced by DwPulA. This contributed to the formation of smaller particles, high colloidal dispersion stability, and increased the absolute value of the zeta potential. Altogether, our findings showed that Deinococcus sp.-derived CBMs can be used to customize GMPs by changing the debranching pattern. • Two pullulanases from Deinococcus raidodurans (DrPulA) and D. wulumuqiensis (DwPulA), which differed in activity only by CBM composition. • CBM b1 from DrPulA had distinct substrate binding coordination. • Incorporation of CBM b1 resulted in a 60% increase in the hydrolysis activity of raw waxy corn starch. • The CBM b1 fusion pullulanase enabled the generation of distinctive α-glucan microparticles with modified characteristics. [ABSTRACT FROM AUTHOR]

The Bacteroides thetaiotaomicron has developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.

Metallothioneins (MTs) are a class of cysteine-rich metal-binding proteins. Cadmium (Cd) is one of the toxic heavy metal pollutants. In our previous research, the full-length cDNA of MT (Cd specificity) from freshwater crab (Sinopotamon henanense) (Sh MT) was cloned and genetically modified to Sh MT3 by site-directed mutagenesis to enhance the tolerance for Cd2+, however, it was limited in actual Cd2+ adsorption due to instability. Here, Sh MT3-CBM, a novel recombinant fusion protein, was prepared. CBM is a carbohydrate binding module that can specifically bind cellulose while Sh MT3 can effectively chelate Cd2+. The biosorbent Cellulose1- Sh MT3-CBM was obtained by screening suitable cellulose materials. The selective adsorption experiments showed that Cellulose1- Sh MT3-CBM had a preference for Cd2+. In low-concentration Cd2+ solutions, the removal efficiency was >99 %, and the adsorption equilibrium was reached within 15 min. The saturated adsorption capacity of Cellulose1- Sh MT3-CBM for Cd2+ is 180.35 ± 4.67 mg/g (Dry Weight). Regeneration experiments showed that adsorption efficiency was maintained after six cycles. The MTT experiment showed that Cellulose1- Sh MT3-CBM had low cytotoxicity. Meanwhile, Cellulose1- Sh MT3-CBM can preferentially remove Cd2+ in actual water samples and boiler sewage. In this study, an environmentally friendly biosorbent which can adsorb Cd2+ efficiently and quickly was prepared for actual water treatment. [Display omitted] • Sh MT3-CBM was obtained by genetic engineering technology. • Cellulose1- Sh MT3-CBM was obtained by immobilization technology. • In low concentration solution, the removal efficiency reaches >99 %. • Cellulose1- Sh MT3-CBM showed excellent reusability. • Cellulose1- Sh MT3-CBM is non-toxic to cells. [ABSTRACT FROM AUTHOR]

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism and binds glycogen, the primary storage form of glucose in liver and skeletal muscle. The effects of disrupting whole-body AMPK-glycogen interactions on exercise capacity and substrate utilization during exercise in vivo remain unknown. We used male whole-body AMPK double knock-in (DKI) mice with chronic disruption of AMPK-glycogen binding to determine the effects of DKI mutation on exercise capacity, patterns of whole-body substrate utilization, and tissue metabolism during exercise. Maximal treadmill running speed and whole-body energy utilization during submaximal running were determined in wild type (WT) and DKI mice. Liver and skeletal muscle glycogen and skeletal muscle AMPK α and β2 subunit content and signaling were assessed in rested and maximally exercised WT and DKI mice. Despite a reduced maximal running speed and exercise time, DKI mice utilized similar absolute amounts of liver and skeletal muscle glycogen compared to WT. DKI skeletal muscle displayed reduced AMPK α and β2 content versus WT, but intact relative AMPK phosphorylation and downstream signaling at rest and following exercise. During submaximal running, DKI mice displayed an increased respiratory exchange ratio, indicative of greater reliance on carbohydrate-based fuels. In summary, whole-body disruption of AMPK-glycogen interactions reduces maximal running capacity and skeletal muscle AMPK α and β2 content and is associated with increased skeletal muscle glycogen utilization. These findings highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and expand AMPK's known roles in exercise and metabolism. [ABSTRACT FROM AUTHOR]

Endoglucanases are increasingly being touted as the ultimate solution for reducing energy consumption during the refining process in the pulp and paper industry. However, due to the high variety of endoglucanases in different enzyme formulations, these perform heterogeneously when applied to different pulps. In this study, the effect of four endoglucanases on softwood and hardwood pulp was studied using confocal laser scanning microscopy (CLSM) after addition of fluorescently labelled carbohydrate binding modules (CBMs). Nuclear magnetic resonance (NMR) analysis and high-performance liquid chromatography quantification of released oligo- and monosaccharides was performed for in-depth mechanistical investigation. Changes in the crystallinity levels caused by enzymatic degradation of amorphous regions were monitored by incubation with two different CBMs from Caldicellulosiruptor bescii and from Thermobifida fusca with high preference to either amorphous or crystalline regions of cellulose, respectively. When dosed at identical activity on the endoglucanase specific CellG5 substrate, CLSM analysis indicated the highest decrease of amorphous regions for those endoglucanases which were also most active in laboratory refining trials and which released highest amounts of cellooligomers from pulp. Using 13C-NMR analysis, an increase in para-crystalline cellulose caused by enzyme application was observed. Release of reducing sugars was determined at identical CellG5 dosage, indicating a high variance between the enzymes, especially when complex enzyme formulations were used. Scanning electron microscopy images were obtained for visualization of the endoglucanase activity. The results of mechanistical studies indicate that reduction of amorphous moieties of pulp by endoglucanases is especially beneficial for the refining process. [ABSTRACT FROM AUTHOR]

There has been significant interest over recent years in the production and application of sustainable and green materials. Among these, nanocellulose has incurred great interest because of its exceptional properties and wide range of potential applications, including in Pickering emulsions. However, the production cost of these cellulosic materials has limited their application. In this study, the capability of a new type of cheaper cellulosic material, cellulose filaments (CFs), in formulating stable oil in water Pickering emulsions was investigated and compared with three conventional nanocelluloses, namely cellulose nanocrystals (CNCs), cellulose nanofibrils (CNFs) and TEMPO-oxidized CNFs (TEMPO-CNFs). Results showed that CFs can provide stable surfactant-free emulsions over wide ranges of salt concentration (0–500 mM) and pH (2–10), as indicated by the near-constant oil droplet size and dewatering index of the emulsions. This is due to the ability of CFs to strongly adsorb to the oil and water interface, as evidenced by Cryo-SEM and visualized through labelled CFs with engineered carbohydrate-binding module (CBM2a) conjugated with green fluorescent protein (CBM2a-eGFP) under fluorescent microscopy. Compared to the emulsions stabilized by other types of nanocelluloses, the CF-stabilized emulsion demonstrated a larger average droplet size and comparable (with CNFs) or better (than CNCs and TEMPO-CNFs) stability, which is partially attributed to the higher viscosity of continuous phase in the presence of CFs. The results of this study demonstrate the use of CFs as a novel and cheaper cellulosic material for stabilizing emulsions, which opens the door to a range of markets from the food industry to engineering applications. [ABSTRACT FROM AUTHOR]

The AMP-activated protein kinase (AMPK) is a central regulator of cellular energy balance and metabolism and binds glycogen, the primary storage form of glucose in liver and skeletal muscle. The effects of disrupting whole-body AMPK-glycogen interactions on exercise capacity and substrate utilization during exercise in vivo remain unknown. We used male whole-body AMPK double knock-in (DKI) mice with chronic disruption of AMPK-glycogen binding to determine the effects of DKI mutation on exercise capacity, patterns of whole-body substrate utilization, and tissue metabolism during exercise. Maximal treadmill running speed and whole-body energy utilization during submaximal running were determined in wild type (WT) and DKI mice. Liver and skeletal muscle glycogen and skeletal muscle AMPK α and β2 subunit content and signaling were assessed in rested and maximally exercised WT and DKI mice. Despite a reduced maximal running speed and exercise time, DKI mice utilized similar absolute amounts of liver and skeletal muscle glycogen compared to WT. DKI skeletal muscle displayed reduced AMPK α and β2 content versus WT, but intact relative AMPK phosphorylation and downstream signaling at rest and following exercise. During submaximal running, DKI mice displayed an increased respiratory exchange ratio, indicative of greater reliance on carbohydrate-based fuels. In summary, whole-body disruption of AMPK-glycogen interactions reduces maximal running capacity and skeletal muscle AMPK α and β2 content and is associated with increased skeletal muscle glycogen utilization. These findings highlight potential unappreciated roles for AMPK in regulating tissue glycogen dynamics and expand AMPK’s known roles in exercise and metabolism.

Mesenchymal stromal cells (MSCs) have the potential to be used as autologous or allogenic cell therapy in several diseases due to their beneficial secretome and capacity for immunomodulation and differentiation. However, clinical trials using MSCs require a large number of cells. As an alternative to traditional culture flasks, suspension bioreactors provide a scalable platform to produce clinically relevant quantities of cells. When cultured in bioreactors, anchorage-dependent cells like MSCs require the addition of microcarriers, which provide a surface for cell attachment while in suspension. The best performing microcarriers are typically coated in animal derived proteins, which increases cellular attachment and proliferation but present issues from a regulatory perspective. To overcome this issue, a recombinant fusion protein was generated linking basic fibroblast growth factor (bFGF) to a cellulose-specific carbohydrate binding module (CBM) and used to functionalize the surface of cellulose microcarriers for the expansion of human umbilical MSCs in suspension bioreactors. The fusion protein was shown to support the growth of MSCs when used as a soluble growth factor in the absence of cellulose, readily bound to cellulose microcarriers in a dose-dependent manner, and ultimately improved the expansion of MSCs when grown in bioreactors using cellulose microcarriers. The use of CBM fusion proteins offers a simple method for the surface immobilization of growth factors to animal component-free substrates such as cellulose, which can be used alongside bioreactors to increase growth factor lifespan, decrease culture medium cost, and increase cell production in the manufacturing of therapeutic cells.

Exoglucanase (CBH) is the rate limiting enzyme in the process of cellulose degradation. The carbohydrate binding module (CBM) can improve the accessibility of cellulase to substrate, thereby promoting the enzymatic hydrolysis of cellulase. In this study, the influence of CBM on the properties of GH6 exoglucanase from Chaetomium thermophilum (CtCBH) is systematically explored from three perspectives: the fusion of exogenous CBM, the exogenous CBM replacement of its own CBM, and the deletion of its own CBM. The parental and reconstructed CtCBH presented the same optimum pH (6.0) and temperature (60 °C) for maximum activity. Fusion of exogenous CBM increased the binding capacity of CtCBH to Avicel by 8% and 9%, respectively, but it had no significant effect on its catalytic activity. The exogenous CBM replacement of its own CBM resulted in a 12% reduction in the binding ability of CtCBH to Avicel, and a 26% reduction in the catalytic activity of Avicel. The deletion of its own CBM significantly reduced the binding ability of CtCBH to Avicel by approximately 53%, but its catalytic activity was not obviously reduced. These observations suggest that binding ability of CBM is not necessary for the catalysis of CtCBH. • CBM is not necessary for the catalysis of CtCBH. • The deletion of CBM significantly reduced the binding ability of CtCBH. • The deletion of CBM did not significantly reduce its catalytic activity. • The fusion of exogenous CBM can improve the binding ability of CtCBH. • The replacement of CBM affected the catalytic activity and binding ability of CtCBH. [ABSTRACT FROM AUTHOR]

BackgroundCarbohydrate binding modules (CBMs) bind polysaccharides and help target glycoside hydrolases catalytic domains to their appropriate carbohydrate substrates. To better understand how CBMs can improve cellulolytic enzyme reactivity, representatives from each of the 18 families of CBM found in Ruminoclostridium thermocellum were fused to the multifunctional GH5 catalytic domain of CelE (Cthe_0797, CelEcc), which can hydrolyze numerous types of polysaccharides including cellulose, mannan, and xylan. Since CelE is a cellulosomal enzyme, none of these fusions to a CBM previously existed.ResultsCelEcc_CBM fusions were assayed for their ability to hydrolyze cellulose, lichenan, xylan, and mannan. Several CelEcc_CBM fusions showed enhanced hydrolytic activity with different substrates relative to the fusion to CBM3a from the cellulosome scaffoldin, which has high affinity for binding to crystalline cellulose. Additional binding studies and quantitative catalysis studies using nanostructure-initiator mass spectrometry (NIMS) were carried out with the CBM3a, CBM6, CBM30, and CBM44 fusion enzymes. In general, and consistent with observations of others, enhanced enzyme reactivity was correlated with moderate binding affinity of the CBM. Numerical analysis of reaction time courses showed that CelEcc_CBM44, a combination of a multifunctional enzyme domain with a CBM having broad binding specificity, gave the fastest rates for hydrolysis of both the hexose and pentose fractions of ionic-liquid pretreated switchgrass.ConclusionWe have shown that fusions of different CBMs to a single multifunctional GH5 catalytic domain can increase its rate of reaction with different pure polysaccharides and with pretreated biomass. This fusion approach, incorporating domains with broad specificity for binding and catalysis, provides a new avenue to improve reactivity of simple combinations of enzymes within the complexity of plant biomass.

Because of their potential impact in food and bioprocessing, diagnostics, green(er) chemistry, and waste remediation, new enzyme immobilization technologies continue to be explored. In this work, a recombinant lactase (β-galactosidase) (LacZ) presenting a carbohydrate binding module (LacZ-CBM), was engineered, expressed, and immobilized onto cellulose (LacZ-CBM:cell). Binding density, activity, and pH and temperature activity profiles were characterized in comparison to wild type lactase (LacZ). LacZ-CBM effectively self-immobilized onto cellulose (Sigmacell®) in less than 2 h, demonstrating that recombinant enzymes with cellulose binding modules can enable immobilization onto solid supports without the need for chemical crosslinking agents. The immobilized recombinant LacZ-CBM:cell retained over 30% of its initial activity over 9 cycles. Both immobilized and non-immobilized LacZ-CBM presented similar optimum reaction conditions as wild type lactase, which demonstrates that the addition of a carbohydrate binding module (CBM) to lactase does not alter its optimum reaction conditions. This work has direct relevance to immobilized lactase applications (e.g. lactose reduced milk; oligosaccharide production) and serves as a model for other greener enzyme immobilization applications. [ABSTRACT FROM AUTHOR]

Alginate lyases are potential agents for disrupting alginate‐rich Pseudomonas biofilms in the infected lungs of cystic fibrosis patients but there is as yet no clinically approved alginate lyase that can be used as a therapeutic. We report here the endolytic alginate lyase activity of a recombinant Cellulophaga algicola alginate lyase domain (CaAly) encoded by a gene that also codes for an N‐terminal carbohydrate‐binding module, CBM6, and a central F‐type lectin domain (CaFLD). CaAly degraded both polyM and polyG alginates with optimal temperature and pH of 37°C and pH 7, respectively, with greater preference for polyG. Recombinant CaFLD bound to fucosylated glycans with a preference for H‐type 2 glycan motif, and did not have any apparent effect on the enzyme activity of the co‐associated alginate lyase domain in the recombinant protein construct, CaFLD_Aly. We assessed the potential of CaAly and other alginate lyases previously reported in published literature to inhibit biofilm formation by a clinical strain, Pseudomonas aeruginosa MCC 2081. Of all the alginate lyases tested, CaAly displayed most inhibition of in vitro biofilm formation on plastic surfaces. We also assessed its inhibitory ability against P. aeruginosa 2081 biofilms formed over a monolayer of A549 lung epithelial cells. Our study indicated that CaAly is efficacious in inhibition of biofilm formation even on A549 lung epithelial cell line monolayers. [ABSTRACT FROM AUTHOR]

Summary: Plants must rearrange the network of complex carbohydrates in their cell walls during normal growth and development. To accomplish this, all plants depend on proteins called expansins that nonenzymatically loosen noncovalent bonding between cellulose microfibrils.Surprisingly, expansin genes have more recently been found in some bacteria and microbial eukaryotes, where their biological functions are largely unknown.Here, we reconstruct a comprehensive phylogeny of microbial expansin genes. We find these genes in all eukaryotic microorganisms that have structural cell wall cellulose, suggesting expansins evolved in ancient marine microorganisms long before the evolution of land plants. We also find expansins in an unexpectedly high diversity of bacteria and fungi that do not have cellulosic cell walls. These bacteria and fungi inhabit varied ecological contexts, mirroring the diversity of terrestrial and aquatic niches where plant and/or algal cellulosic cell walls are present.The microbial expansin phylogeny shows evidence of multiple horizontal gene transfer events within and between bacterial and eukaryotic microbial lineages, which may in part underlie their unusually broad phylogenetic distribution. Overall, expansins are unexpectedly widespread in bacteria and eukaryotes, and the contribution of these genes to microbial ecological interactions with plants and algae has probbaly been underappreciated. [ABSTRACT FROM AUTHOR]

The specific interaction between carbohydrate binding modules (CBMs) and substrates is of utmost importance due to it affects the biological activity of the parent enzymes and determines the chemo-mechanical properties of protein-cellulose composites. In this investigation, a quartz crystal microbalance with dissipation monitoring was employed to study, in situ and in real-time, the adsorption behaviors, conformational changes and associated thermodynamics that define the specific interactions between type A CBMs (CBM1 and CBM3) and cellulose (crystalline, CNC and nanofibrillated, CNF). CBM1 and CBM3 specifically bind to CNC and CNF substrates, with the CBM3 forming more rigid adsorbed layers at 25 °C. Despite the wide variation in adsorption enthalpy (ΔH) and entropy (ΔS), depending on the experimental conditions, a negative Gibbs free energy (ΔG) was determined from the adsorption isotherms for both CBMs. Particularly, the ΔH and ΔS associated with CBM3 adsorbing on CNF exhibited significant greater values than others due to cellulose chain disorder when swelling. The results further our understandings on the interactions between type A CBMs and cellulose substrates. [ABSTRACT FROM AUTHOR]

To understand the influence of family 3 Carbohydrate Binding Module (hereafter CBM3), single (GH5 cellulase; CelB, CelBΔCBM), bi-chimeric [GH26 endo-mannanase (ManB-1601) and GH11 endo-xylanase (XynB); ManB-XynB [1] , ManB-XynB-CBM] and tri-chimeric [ManB-XynB-CelB [1] , ManB-XynB-CelBΔCBM] enzyme variants (fused or deleted of CBM) were produced and purified to homogeneity. CBM3 did not alter the pH and temperature optima of bi- and tri-chimeric enzymes but improved the pH and temperature stability of ManB in CBM variants of bi-/tri-chimeric enzymes. Truncation of CBM in CelB shifted the pH optimum and increased the melting temperature (T m 65 ℃). CBM3 improved both substrate affinity (K m) and catalytic efficiency (k cat /K m) of fused enzymes in tri-chimera and CelB but only K m for bi-chimera. Far-UV CD of CelB and bi- and tri-chimeric enzymes suggested that CBM3 improved the α-helical content and compactness in the native state but did not prevent disintegration of secondary structural contents at acidic pH. Steady-state fluorescence studies suggested that under acidic conditions CBM3 prevented the exposure of hydrophobic patches in bi-chimeric protein but could not avert the opening up of chimeric enzyme structure. Aqueous enzyme assisted treatment of mature coconut kernel using single, bi- and tri-chimeric enzymes led to cracks, peeling and fracturing of the matrix and improved the oil yield by up to 22%. • Functional expression of CBM3 variants of single and bi- and tri-chimeric enzymes. • Fusion or deletion of CBM3 alters biochemical properties of enzymes. • CBM3 renders higher structural compactness in enzymes under native state. • CBM3 does not prevent denaturation of enzymes at acidic pH. • Bi-chimera fused with CBM3 improves oil extraction from mature coconut by 22%. [ABSTRACT FROM AUTHOR]

• CDH of Termitomyces clypeatus possesses distinct carbohydrate binding module. • First report of three domains- cytochrome, dehydrogenase, CBM in basidiomycete CDH. • Dehydrogenase domain is devoid of cellulose binding residues. • Phylogenetic analysis clusters it with the CDH of ascomycetes group of fungi. • Possible phylogenetic status of this fungus have been discussed. Filamentous fungi secrete various oxidative enzymes to degrade the glycosidic bonds of polysaccharides. Cellobiose dehydrogenase (CDH) (E.C.1.1.99.18) is one of the important lignocellulose degrading enzymes produced by various filamentous fungi. It contains two stereo specific ligand binding domains, cytochrome and dehydrogenase - one for heme and the other for flavin adenine dinucleotide (FAD) respectively. The enzyme is of commercial importance for its use in amperometric biosensor, biofuel production, lactose determination in food, bioremediation etc. Termitomyces clypeatus , an edible fungus belonging to the basidiomycetes group, is a good producer of CDH. In this paper we have analyzed the structural properties of this enzyme from T. clypeatus and identified a distinct carbohydrate binding module (CBM) which is not present in most fungi belonging to the basidiomycetes group. In addition, the dehydrogenase domain of T. clypeatus CDH exhibited the absence of cellulose binding residues which is in contrast to the dehydrogenase domains of CDH of other basidiomycetes. Sequence analysis of cytochrome domain showed that the important residues of this domain were conserved like in other fungal CDHs. Phylogenetic tree, constructed using basidiomycetes and ascomycetes CDH sequences, has shown that very surprisingly the CDH from T. clypeatus, which is classified as a basidiomycetes fungus, is clustered with the ascomycetes group. A homology model of this protein has been constructed using the CDH enzyme of ascomycetes fungus Myricoccum thermophilum as a template since it has been found to be the best match sequence with T. clypeatus CDH. We also have modelled the protein with its substrate, cellobiose, which has helped us to identify the substrate interacting residues (L354, P606, T629, R631, Y649, N732, H733 and N781) localized within its dehydrogenase domain. Our computational investigation revealed for the first time the presence of all three domains - cytochrome, dehydrogenase and CBM - in the CDH of T. clypeatus, a basidiomycetes fungus. In addition to discovering the unique structural attributes of this enzyme from T. clypeatus , our study also discusses the possible phylogenetic status of this fungus. [ABSTRACT FROM AUTHOR]

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis.

Biosynthesis of starch is catalyzed by a cascade of enzymes. The activity of a large number of these enzymes depends on interaction with polymeric substrates via carbohydrate binding sites, which are situated outside of the catalytic site and its immediate surroundings including the substrate-binding crevice. Such secondary binding sites can belong to distinct starch binding domains (SBDs), classified as carbohydrate binding modules (CBMs), or be surface binding sites (SBSs) exposed on the surface of catalytic domains. Currently in the Carbohydrate-Active enZYmes (CAZy) database SBDs are found in 13 CBM families. Four of these families; CBM20, CBM45, CBM48, and CBM53 are represented in enzymes involved in starch biosynthesis, namely starch synthases, branching enzymes, isoamylases, glucan, water dikinases, and α-glucan phosphatases. A critical role of the SBD in activity has not been demonstrated for any of these enzymes. Among the well-characterized SBDs important for starch biosynthesis are three CBM53s of Arabidopsis thaliana starch synthase III, which have modest affinity. SBSs, which are overall less widespread than SBDs, have been reported in some branching enzymes, isoamylases, synthases, phosphatases, and phosphorylases active in starch biosynthesis. SBSs appear to exert roles similar to CBMs. SBSs, however, have also been shown to modulate specificity for example by discriminating the length of chains transferred by branching enzymes. Notably, the difference in rate of occurrence between SBDs and SBSs may be due to lack of awareness of SBSs. Thus, SBSs as opposed to CBMs are not recognized at the protein sequence level, which hampers their identification. Moreover, only a few SBSs in enzymes involved in starch biosynthesis have been functionally characterized, typically by structure-guided site-directed mutagenesis. The glucan phosphatase Like SEX4 2 from A. thaliana has two SBSs with weak affinity for β-cyclodextrin, amylose and amylopectin, which were indicated by mutational analysis to be more important than the active site for initial substrate recognition. The present review provides an update on occurrence of functional SBDs and SBSs in enzymes involved in starch biosynthesis. [ABSTRACT FROM AUTHOR]

Carbohydrate Binding Modules (CBMs) targeting cellulose, xylan and mannan have been reported, however, a CBM targeting rhamnogalacturonan I (RG I) has never been identified. We had studied earlier a rhamnogalacturonan lyase ( Ct RGL) from Clostridium thermocellum that was associated with a family 35 CBM, Rgl- CBM35. In this study we show that Rgl -CBM35 displays binding with β- d -glucuronic acid (β-D-Glc p A), Δ4,5-anhydro- d -galactopyranosyluronic acid (Δ4,5-Gal p A), rhamnogalacturonan I, arabinan, galactan, glucuronoxylans and arabinoxylans. Rgl -CBM35 contains a conserved ligand binding site in the loops known for binding β-D-Glc p A and Δ4,5-Gal p A moiety of unsaturated RG I and pectic-oligosaccharides. Mutagenesis revealed that Asn118 plays an important role in binding β-D-Glc p A, Δ4,5-Gal p A, sugarbeet arabinan and potato galactan at its conserved ligand binding site present in surface exposed loops. EDTA-treated Rgl -CBM35 showed no affinity towards β-D-Glc p A and Δ4,5-Gal p A underscoring Ca 2+ mediated ligand recognition. Contrastingly, the EDTA-treated Rgl -CBM35 and its mutant N118A displayed affinity for sugarbeet arabinan and potato galactan. The curtailed affinity of Y37A/N118A and R69A/N118A double mutants towards sugarbeet arabinan emphasized the presence of a second ligand binding site. Rgl -CBM35 is the first CBM reported to primarily target RG I and also is the first member of family 35 CBM possessing at least two ligand binding sites. [ABSTRACT FROM AUTHOR]

Enzymatically-active bacterial cellulose (BC) was prepared by non-covalent immobilization of a hybrid enzyme composed by a β-galactosidase from Thermotoga maritima (TmLac) and a carbohydrate binding module (CBM2) from Pyrococcus furiosus . TmLac-CBM2 protein was bound to BC, with higher affinity at pH 6.5 than at pH 8.5 and with high specificity compared to the non-engineered enzyme. Both hydrated (HBC) and freeze-dried (DBC) bacterial cellulose showed equivalent enzyme binding efficiencies. Initial reaction rate of HBC-bound enzyme was higher than DBC-bound and both of them were lower than the free enzyme. However, enzyme performance was similar in all three cases for the hydrolysis of 5% lactose to a high extent. Reuse of the immobilized enzyme was limited by the stability of the β-galactosidase module, whereas the CBM2 module provided stable attachment of the hybrid enzyme to the BC support, after long incubation periods (3 h) at 75 °C. [ABSTRACT FROM AUTHOR]

Bifunctional cellulase (glycoside hydrolase 5, GH5) from Bacillus sp. D04 having both endo- and exoglucanase activities was fused with two types of carbohydrate binding modules (CBMs). CBM3 from Bacillus sp. D04 and CBM9 from Thermotoga maritima Xyn10A were added to GH5 to hydrolyze microcrystalline cellulose (Avicel) as well as water-soluble cellulose (carboxymethyl cellulose, CMC). The optimum temperature of GH5 was 50oC, while it increased to 60oC for the fusion GH5-CBM3 and GH5-CBM9, indicating that addition of CBM increased the thermostability of the enzyme. Addition of CBM3 and CBM9 enhanced the GH5 affinity (KM), for which KM decreased from 104 to 33.9 ~ 35.1 mg/mL for CMC, and from 115 to 55.5 ~ 80.3 mg/mL for Avicel, respectively. The catalytic efficiency (kcat/KM) also increased from 4.80 to 5.36 ~ 6.46 (mL/mg)/sec for CMC, and from 1.77 to 2.40 ~ 4.45 (mL/mg)/sec for Avicel, respectively, by addition of CBM3 and CBM9. [ABSTRACT FROM AUTHOR]

Enzymatic processing is considered a promising approach for advancing environmentally friendly industrial processes, such as the use of endoglucanase (EG) enzyme in the production of nanocellulose. However, there is ongoing debate regarding the specific properties that make EG pretreatment effective in isolating fibrillated cellulose. To address this issue, we investigated EGs from four glycosyl hydrolase (GH) families (5, 6, 7, and 12) and examined the roles of the three-dimensional structure and catalytic features, with a focus on the presence of a carbohydrate binding module (CBM). Using eucalyptus Kraft wood fibers, we produced cellulose nanofibrils (CNFs) through mild enzymatic pretreatment, followed by disc ultra-refining. Comparing the results with the control (without pretreatment), we observed that GH5 and GH12 enzymes (without CBM) reduced fibrillation energy by approximately 15 %. The most significant energy reduction, 25 and 32 %, was achieved with GH5 and GH6 linked to CBM, respectively. Notably, these CBM-linked EGs improved the rheological properties of CNF suspensions without releasing soluble products. In contrast, GH7-CBM exhibited significant hydrolytic activity, resulting in the release of soluble products, but did not contribute to a reduction in fibrillation energy. This discrepancy can be attributed to the large molecular weight and wide cleft of GH7-CBM, which led to the release of soluble sugars but had little impact on fibrillation. Our findings suggest that the improved fibrillation observed with EG pretreatment is primarily driven by efficient enzyme adsorption on the substrate and modification of the surface viscoelasticity (amorphogenesis), rather than hydrolytic activity or release of products. Energy consumption and properties of cellulose nanofibers produced after single-step, low-loading enzyme pretreatment of wood pulp. [Display omitted] [ABSTRACT FROM AUTHOR]

A recombinant β-1,4 endoglucanase, At GH9C-CBM3A-CBM3B from Acetivibrio thermocellus ATCC27405 was explored for biochemical properties and the role of its associated CBMs in catalysis. The gene expressing full-length multi-modular β-1,4-endoglucanase (At GH9C-CBM3A-CBM3B) and its truncated derivatives (At GH9C-CBM3A, At GH9C, CBM3A and CBM3B) were independently cloned and expressed in Escherichia coli BL21(DE3) cells and purified. At GH9C-CBM3A-CBM3B showed maximal activity at 55 °C and pH 7.5. At GH9C-CBM3A-CBM3B exhibited highest activity against carboxy methyl cellulose (58.8 U/mg) followed by lichenan (44.5 U/mg), β-glucan (36.2 U/mg) and hydroxy ethyl cellulose (17.9 U/mg). Catalytic module, At GH9C showed insignificant activity against the substrates, signifying the essential requirement of CBMs in catalysis. At GH9C-CBM3A-CBM3B displayed stability in pH range, 6.0–9.0 and thermostability up to 60 °C for 90 min with unfolding transition midpoint (T m) of 65 °C. The generation of cellotetraose and other higher oligosaccharides by At GH9C-CBM3A-CBM3B confirmed it as an endo -β-1,4-glucanase. At GH9C activity was partially recovered by the addition of equimolar concentration of CBM3A, CBM3B or CBM3A + CBM3B by 47 %, 13 % or 50 %, respectively. Moreover, the associated CBMs imparted thermostability to the catalytic module, At GH9C. These results showed that the physical association of At GH9C with its associated CBMs and the cross-talk between CBMs are necessary for At GH9C-CBM3A-CBM3B in effective cellulose catalysis. • At GH9C-CBM3A-CBM3B of Acetivibrio thermocellus is a new endoglucanase of GH9 family • Endoglucanase At GH9C-CBM3A-CBM3B gave maximum activity at 55 °C and pH 7.5 • At GH9C-CBM3A-CBM3B is thermostable and stable in wide range of pH. • Mg2+ ion is present in At GH9C-CBM3A-CBM3B helping in thermostability and catalysis • Physical association of At GH9C with CBMs is needed for effective glucan hydrolysis [ABSTRACT FROM AUTHOR]

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

The expression of cell-wall-targeted Carbohydrate Binding Modules (CBMs) can alter cell wall properties and modulate growth and development in plants such as tobacco and potato. CBM2a identified in xylanase 10A from Cellulomonas fimi is of particular interest for its ability to bind crystalline cellulose. However, its potential for promoting plant growth has not been explored. In this work, we tested the ability of CBM2a to promote growth when expressed using both CaMV35S and a vascular tissue-specific promoter derived from Arabidopsis expansin4 ( AtEXP4) in three plant species: Arabidopsis, Nicotiana tabacum and Eucalyptus camaldulensis. In Arabidopsis, the expression of AtEXP4pro:CBM2a showed trends for growth promoting effects including the increase of root and hypocotyl lengths and the enlargements of the vascular xylem area, fiber cells and vessel cells. However, in N. tabacum, the expression of CBM2a under the control of either CaMV35S or AtEXP4 promoter resulted in subtle changes in the plant growth, and the thickness of secondary xylem and vessel and fiber cell sizes were generally reduced in the transgenic lines with AtEXP4pro:CBM2a. In Eucalyptus, while transgenics expressing CaMV35S:CBM2a showed very subtle changes compared to wild type, those transgenics with AtEXP4pro:CBM2a showed increases in plant height, enlargement of xylem areas and xylem fiber and vessel cells. These data provide comparative effects of expressing CBM2a protein in different plant species, and this finding can be applied for plant biomass improvement. [ABSTRACT FROM AUTHOR]

Physarum polycephalum is an unusual macroscopic myxomycete expressing a large range of glycosyl hydrolases. Among them, enzymes from the GH18 family can hydrolyze chitin, an important structural component of the cell walls in fungi and in the exoskeleton of insects and crustaceans. Low stringency sequence signature search in transcriptomes was used to identify GH18 sequences related to chitinases. Identified sequences were expressed in E. coli and corresponding structures modelled. Synthetic substrates and in some cases colloidal chitin were used to characterize activities. Catalytically functional hits were sorted and their predicted structures compared. All share the TIM barrel structure of the GH18 chitinase catalytic domain, optionally fused to binding motifs, such as CBM50, CBM18, and CBM14, involved in sugar recognition. Assessment of the enzymatic activities following deletion of the C-terminal CBM14 domain of the most active clone evidenced a significant contribution of this extension to the chitinase activity. A classification based on module organization, functional and structural criteria of characterized enzymes was proposed. Physarum polycephalum sequences encompassing a chitinase like GH18 signature share a modular structure involving a structurally conserved catalytic TIM barrels decorated or not by a chitin insertion domain and optionally surrounded by additional sugar binding domains. One of them plays a clear role in enhancing activities toward natural chitin. Myxomycete enzymes are currently poorly characterized and constitute a potential source for new catalysts. Among them glycosyl hydrolases have a strong potential for valorization of industrial waste as well as in therapeutic field. [Display omitted] • Slime mold P. polycephalum expresses a large number of GH18 glycoside hydrolases. • Sequences share decorated variants of the catalytic TIM barrel fold of chitinases. • Sugar binding motifs, such as CBM50, CBM18, and CBM14 are fused to the TIM barrel. • Three of the characterized enzymes efficiently hydrolyze colloidal chitin. • Functional roles of C-and N-terminal extensions were characterized and a classification proposed. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase 9 superfamily, mainly comprising the endoglucanases, and is represented in all three domains of life. The current division of GH9 enzymes, into three subclasses, namely A, B, and C, is centered on parameters derived from sequence information alone. However, this classification is ambiguous, and is limited by the paralogous ancestry of classes B and C endoglucanases, and paucity of biochemical and structural data. Here, we extend this classification schema to putative GH9 endoglucanases present in green plants, with an emphasis on identifying novel members of the class C subset. These enzymes cleave the β(1→4) linkage between non-terminal adjacent D-glucopyranose residues, in both, amorphous and crystalline regions of cellulose. We utilized non redundant plant GH9 enzymes with characterized molecular data, as the training set to construct Hidden Markov Models (HMMs) and train an Artificial Neural Network (ANN). The parameters that were used for predicting dominant enzyme function, were derived from this training set, and subsequently refined on 147 sequences with available expression data. Our knowledge-based approach, can ascribe differential endoglucanase activity (A, B, or C) to a query sequence with high confidence, and was used to construct a local repository of class C GH9 endoglucanases (GH9C=241) from 32 sequenced green plants.

Two novel glycosyl hydrolase family 5 (GH5) β-mannanases (AoMan5A and AoMan5B) were identified from Aspergillus oryzae RIB40 by genome mining. The AoMan5A contains a predicted family 1 carbohydrate binding module (CBM-1), located at its N-terminal. The AoMan5A, AoMan5 B and truncated mutant AoMan5AΔCL (truncating the N-terminal CBM and linker of AoMan5A) were expressed retaining the N-terminus of the native protein in Pichia pastoris GS115 by pPIC9 K M . The specific enzyme activity of the purified reAoMan5A, reAoMan5 B and reAoMan5AΔCL towards locust bean gum at pH 3.6 and 40 °C for 10 min, was 8.3, 104.2 and 15.8 U/mg, respectively. The temperature properties of the reAoMan5AΔCL were improved by truncating CBM. They can degrade the pretreated konjac flour and produce prebiotics. In addition, they had excellent stability under simulative gastric fluid and simulative prilling process. All these properties make these recombinant β-mannanases potential additives for use in the food and feed industries. [ABSTRACT FROM AUTHOR]

The model 3-D structure of xylanase KRICT PX3 (JF320814) identified by DNA sequence analysis revealed a catalytic domain and CBM4-9 which functions as a xylan binding domain (XBD). To identify its role in xylan hydrolysis, six expression plasmids were constructed encoding the N-terminal CBM plus the catalytic domain or different glycosyl hydrolases, and the biochemical properties of the recombinant enzymes were compared to the original structure of PX3 xylanase. All six of the recombinant xylanases with the addition of CBM in the pIVEX-GST expression vector showed no improved PX3 hydrolytic activity. However, the absence of the CBM domain resulted in a decrement of 40% in thermostability, movement of the optimal temperature from 55 °C to 45 °C, alteration of the optimal pH range from 5–10 to 6–8, and reduction of the enzymatic activity to one-second under the same condition, respectively. The putative XBD in PX3 comprises a new N-terminal domain homologous to the catalytic thermostabilizing domains from other xylanases. Analysis of the main products released from xylan indicate that the recombinant enzymes act as endo -1,4-β-xylanases but differ in their hydrolysis of xylan from beech wood, birch wood, and oat spelt. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase 9 superfamily, mainly comprising the endoglucanases, is represented in all three domains of life. The current division of GH9 enzymes, into three subclasses, namely A, B, and C, is centered on parameters derived from sequence information alone. However, this classification is ambiguous, and is limited by the paralogous ancestry of classes B and C endoglucanases, and paucity of biochemical and structural data. Here, we extend this classification schema to putative GH9 endoglucanases present in green plants, with an emphasis on identifying novel members of the class C subset. These enzymes cleave the β(1 → 4) linkage between non-terminal adjacent D-glucopyranose residues, in both, amorphous and crystalline regions of cellulose. We utilized non redundant plant GH9 enzymes with characterized molecular data, as the training set to construct Hidden Markov Models (HMMs) and train an Artificial Neural Network (ANN). The parameters that were used for predicting dominant enzyme function, were derived from this training set, and subsequently refined on 147 sequences with available expression data. Our knowledge-based approach, can ascribe differential endoglucanase activity (A, B, or C) to a query sequence with high confidence, and was used to construct a local repository of class C GH9 endoglucanases (GH9C = 241) from 32 sequenced green plants. [ABSTRACT FROM AUTHOR]

The heteropolysaccharide xyloglucan (XyG) comprises up to one-quarter of the total carbohydrate content of terrestrial plant cell walls and, as such, represents a significant reservoir in the global carbon cycle. The complex composition of XyG requires a consortium of backbone-cleaving endo-xyloglucanases and side-chain cleaving exo-glycosidases for complete saccharification. The biochemical basis for XyG utilization by the model Gram-negative soil saprophytic bacterium Cellvibrio japonicus is incompletely understood, despite the recent characterization of associated side-chain cleaving exo-glycosidases. We present a detailed functional and structural characterization of a multimodular enzyme encoded by gene locus CJA_2477. The CJA_2477 gene product comprises an N-terminal glycoside hydrolase family 74 ( GH74) endo-xyloglucanase module in train with two carbohydrate-binding modules ( CBMs) from families 10 and 2 ( CBM10 and CBM2). The GH74 catalytic domain generates Glc4-based xylogluco-oligosaccharide (Xy GO) substrates for downstream enzymes through an endo-dissociative mode of action. X-ray crystallography of the GH74 module, alone and in complex with Xy GO products spanning the entire active site, revealed a broad substrate-binding cleft specifically adapted to XyG recognition, which is composed of two seven-bladed propeller domains characteristic of the GH74 family. The appended CBM10 and CBM2 members notably did not bind XyG, nor other soluble polysaccharides, and instead were specific cellulose-binding modules. Taken together, these data shed light on the first step of xyloglucan utilization by C. japonicus and expand the repertoire of GHs and CBMs for selective biomass analysis and utilization. Database Structural data have been deposited in the RCSB protein database under the Protein Data Bank codes: , , and . [ABSTRACT FROM AUTHOR]

Background Galactose oxidase (GaO) selectively oxidizes the primary hydroxyl of galactose to a carbonyl, facilitating targeted chemical derivatization of galactose-containing polysaccharides, leading to renewable polymers with tailored physical and chemical properties. Here we investigate the impact of a family 29 glucomannan binding module on the activity and binding of GaO towards various polysaccharides. Specifically, CBM29-1-2 from Piromyces equi was separately linked to the N- and C-termini of GaO. Results Both GaO–CBM29 and CBM29–GaO were successfully expressed in Pichia pastoris , and demonstrated enhanced binding to galactomannan, galactoglucomannan and galactoxyloglucan. The position of the CBM29 fusion affected the enzyme function. Particularly, C-terminal fusion led to greatest increases in galactomannan binding and catalytic efficiency, where relative to wild-type GaO, k cat / K m values increased by 7.5 and 19.8 times on guar galactomannan and locust bean galactomannan, respectively. The fusion of CBM29 also induced oligomerization of GaO–CBM29. Major conclusions Similar to impacts of cellulose-binding modules associated with cellulolytic enzymes, increased substrate binding impeded the action of GaO fusions on more concentrated preparations of galactomannan, galactoglucomannan and galactoxyloglucan; this was especially true for GaO–CBM29. Given the N-terminal positioning of the native galactose-binding CBM32 in GaO, the varying impacts of N-terminal versus C-terminal fusion of CBM29-1-2 may reflect competing action of neighboring CBMs. General significance This study thoroughly examines and discusses the effects of CBM fusion to non-lignocellulytic enzymes on soluble polysaccharides. Herein kinetics of GaO on galactose containing polysaccharides is presented for the first time. [ABSTRACT FROM AUTHOR]

Filter paper provides an excellent matrix for retention of proteins containing a cellulose binding domain. To use this capability for manipulating recombinant fusion proteins, binding and elution parameters were explored and procedures developed for small scale purification, modification and assay. Proteins were tagged with the cellulose binding domain from the Clostridium thermocellum CipB gene via a cleavable linker. Filter paper disks of 6 mm diameter were able to bind up to 80 μg protein although there was a substantial dependence on molecular size. Different means of introducing fusion proteins to the disks allow either binding within 20 min from microliter volumes or slower binding from milliliter volumes. Elution with protease in small volumes yielded greater than 10 μg amounts with concentrations in the 1–2 mg/ml range. To demonstrate their utility, disks were used for small scale protein purification, covalent modification of protein, immunoprecipitation, and in a binding assay. These versatile methods allow parallel processing of multiple samples and may find many uses when only small amounts of protein are needed. [Display omitted] • Rapid, high throughput, small scale purification of recombinant fusion proteins. • Binding assays on filter paper disks. • Protein modification on filter paper disks. [ABSTRACT FROM AUTHOR]