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Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations. The results reveal that BlCel9A breaks down cellulosic substrates, releasing cellobiose and glucose as the major products, but is highly inefficient in cleaving oligosaccharides shorter than cellotetraose. In addition, fungal lytic polysaccharide oxygenase (LPMO) TtLPMO9H enhances depolymerization of crystalline cellulose by BlCel9A, while exhibiting minimal impact on amorphous cellulose. The crystal structures of BlCel9A in both apo form and bound to cellotriose and cellohexaose were elucidated, unveiling the interactions of BlCel9A with the ligands and their contribution to substrate binding and products release. MD simulation analysis reveals that BlCel9A exhibits higher interdomain flexibility under acidic conditions, and SAXS experiments indicate that the enzyme flexibility is induced by pH and/or temperature. Our findings provide new insights into BlCel9A substrate specificity and binding, and synergy with the LPMOs.

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Carbohydrate-binding modules (CBMs) constitute independently folded domains typically associated with carbohydrate-active enzymes (CAZymes). These modules are considered to have a rigid structure without notable conformational changes upon ligand binding, exhibiting a complementary topography in relation to the target carbohydrate. Herein, the high-resolution SAD-solved structure of a CBM from family 3 (BsCBM3) that binds to crystalline cellulose is reported in two crystalline forms. This module showed molecular plasticity with structural differences detected between the two crystalline forms and high RMSD values when compared to NMR ensemble of models. Pronounced structural variances were observed in the cellulose binding interface between NMR and XTAL structures, which were corroborated by molecular dynamics simulations. These findings support that family 3 CBMs targeting to cellulose are rather structurally dynamic modules than rigid entities, suggesting a potential role of conformational changes in polysaccharide recognition and modulation of enzyme activity.

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The ß-d-glucans are abundant cell wall polysaccharides in many cereals and contain both (1,3)- and (1,4)-bonds. The ß-1,3-1,4-glucanases (EC 3.2.1.73) hydrolyze ß-(1,4)-d-glucosidic linkages in glucans, and have applications in both animal and human food industries. A chimera between the family 11 carbohydrate-binding module from Ruminoclostridium (Clostridium)thermocellumcelH (RtCBM11), with the ß-1,3-1,4-glucanase from Bacillus subtilis (BglS) was constructed by end-to-end fusion (RtCBM11-BglS) to evaluate the effects on the catalytic function and its application in barley ß-glucan degradation for the brewing industry. The parental and chimeric BglS presented the same optimum pH (6.0) and temperature (50 °C) for maximum activity. The RtCBM11-BglS showed increased thermal stability and 30% higher hydrolytic efficiency against purified barley ß-glucan, and the rate of hydrolysis of ß-1,3-1,4-glucan in crude barley extracts was significantly increased. The enhanced catalytic performance of the RtCBM11-BglS may be useful for the treatment of crude barley extracts in the brewing industry.

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Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed ß-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 ß-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design.

RESUMO

We analyzed the structure to function relationships in Ruminococcus albus 8 xylanase 10A (RalXyn10A) finding that the N-terminus 34-amino acids sequence (N34) in the protein is particularly functional. We performed the recombinant wild type enzyme's characterization and that of the truncated mutant lacking the N34 extreme (RalΔN34Xyn10A). The truncated enzyme exhibited about half of the activity and reduced affinity for binding to insoluble saccharides. These suggest a (CBM)-like function for the N34 motif. Besides, RalXyn10A activity was diminished by redox agent dithiothreitol, a characteristic absent in RalΔN34Xyn10A. The N34 sequence exhibited a significant similarity with protein components of the ABC transporter of the bacterial membrane, and this motif is present in other proteins of R. albus 8. Data suggest that N34 would confer RalXyn10A the capacity to interact with polysaccharides and components of the cell membrane, enhancing the degradation of the substrate and uptake of the products by the bacterium.

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Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data. BlCel9 is an endoglucanase exhibiting maximum catalytic efficiency at pH 7.0 and 60 °C. Furthermore, it retains 80% of catalytic activity within a broad range of pH values (5.5-8.5) and temperatures (up to 50 °C) for extended periods of time (over 48 h). It exhibits the highest hydrolytic activity against phosphoric acid swollen cellulose (PASC), followed by bacterial cellulose (BC), filter paper (FP), and to a lesser extent carboxymethylcellulose (CMC). The HPAEC-PAD analysis of the hydrolytic products demonstrated that the end product of the enzymatic hydrolysis is primarily cellobiose, and also small amounts of glucose, cellotriose, and cellotetraose are produced. SAXS data analysis revealed that the enzyme adopts a monomeric state in solution and has a molecular mass of 65.8 kDa as estimated from SAXS data. The BlCel9 has an elongated shape composed of an N-terminal family 3 carbohydrate-binding module (CBM3c) and a C-terminal GH9 catalytic domain joined together by 20 amino acid residue long linker peptides. The domains are closely juxtaposed in an extended conformation and form a relatively rigid structure in solution, indicating that the interactions between the CBM3c and GH9 catalytic domains might play a key role in cooperative cellulose biomass recognition and hydrolysis.

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Background: Endoglucanase, one of three type cellulases, can randomly cleave internal p-1,4-linkages in cellulose polymers. Thus, it could be applied in agricultural and industrial processes. Results: A novel endoglucanase gene (JqCel5A) was cloned from Jonesia quinghaiensis and functionally expressed in Escherichia coli Rosetta (DE3). It contained 1722 bp and encoded a 573-residue polypeptide consisting of a catalytic domain of glycoside hydrolase family 5 (GH5) and a type 2 carbohydrate-binding module (CBM2), together with a predicted molecular mass of 61.79 kD. The purified JqCel5A displayed maximum activity at 55°C and pH 7.0, with 21.7 U/mg, 26.19 U/mg and 4.81 U/mg towards the substrate carboxymethyl cellulose, barley glucan and filter paper, respectively. Interestingly, JqCel5A exhibited high pH stability over a broad pH range of pH (3-11), and had good tolerance to a wide variety of deleterious chemicals including heavy metals and detergent. The catalytic mechanism of JqCel5A was also investigated by site mutagenesis and homology-modeling in this study. Conclusions: It was believed that these properties might make JqCel5A to be potentially used in the suitable industrial catalytic condition, which has a broad pH fluctuation and/or chemical disturbance.

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α-L-arabinofuranosidases (EC 3.2.1.55) are enzymes involved in the catabolism of several cell-wall polysaccharides such as pectins and hemicelluloses, catalyzing the hydrolysis of terminal non-reducing α-L-arabinofuranosil residues. Bioinformatic analysis of the aminoacidic sequences of Fragaria x ananassa α-L-arabinofuranosidases predict a putative carbohydrate-binding-module of the family CBM_4_9, associated to a wide range of carbohydrate affinities. In this study, we report the characterization of the binding affinity profile to different cell wall polysaccharides of the putative CBM of α-L-arabinofuranosidase 1 from Fragaria x ananassa (CBM-FaARA1). The sequence encoding for the putative CBM was cloned and expressed in Escherichia coli, and the resultant recombinant protein was purified from inclusion bodies by a Nickel affinity chromatography under denaturing conditions. The refolded recombinant protein was then subjected to binding assays and affinity gel electrophoresis, which indicated its ability to bind cellulose and also high affinity for homogalacturonans.

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In recent years, biofuels have attracted great interest as a source of renewable energy owing to the growing global demand for energy, the dependence on fossil fuels, limited natural resources and environmental pollution. However, the cost-effective production of biofuels from plant biomass is still a challenge. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases to polysaccharides, is crucial for enzyme development. Aiming at the structural and functional characterization of novel CBMs involved in plant polysaccharide deconstruction, an analysis of the CAZy database was performed and CBM family 64 was chosen owing to its capacity to bind with high specificity to microcrystalline cellulose and to the fact that is found in thermophilic microorganisms. In this communication, the CBM-encoding module named StX was expressed, purified and crystallized, and X-ray diffraction data were collected from native and derivatized crystals to 1.8 and 2.0 Šresolution, respectively. The crystals, which were obtained by the hanging-drop vapour-diffusion method, belonged to space group P3121, with unit-cell parameters a = b = 43.42, c = 100.96 Šfor the native form. The phases were found using the single-wavelength anomalous diffraction method.

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In recent years, owing to the growing global demand for energy, dependence on fossil fuels, limited natural resources and environmental pollution, biofuels have attracted great interest as a source of renewable energy. However, the production of biofuels from plant biomass is still considered to be an expensive technology. In this context, the study of carbohydrate-binding modules (CBMs), which are involved in guiding the catalytic domains of glycoside hydrolases for polysaccharide degradation, is attracting growing attention. Aiming at the identification of new CBMs, a sugarcane soil metagenomic library was analyzed and an uncharacterized CBM (CBM_E1) was identified. In this study, CBM_E1 was expressed, purified and crystallized. X-ray diffraction data were collected to 1.95 Šresolution. The crystals, which were obtained by the sitting-drop vapour-diffusion method, belonged to space group I23, with unit-cell parameters a = b = c = 88.07 Å.

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Cellulose degrading enzymes usually have a two-domain structure consisting of a catalytic domain and a non-catalytic carbohydrate-binding module. Although it is well known the importance of those modules in cell wall degrading process, their function is not yet fully understood. Here, we analyze the cellulose-hydrolysis activity enhancement promoted by the cellobiohydrolase I carbohydrate-binding module from Trichoderma harzianum. It was cloned, expressed, purified and used in combination with either a commercial cellulase preparation, T. reesei cellobiohydrolase I or its separate catalytic domain to hydrolyze filter paper. In all cases the amount of glucose released was increased, reaching up to 30% gain when the carbohydrate-binding module was added to the reaction. We also show that this effect seems to be mediated by a decrease in the recalcitrance of the cellulosic substrate. This effect was observed both for crystalline cellulose samples which underwent incubation with the CBM prior to application of cellulases and for the ones incubated simultaneously. Our studies demonstrate that family 1 carbohydrate-binding modules are able to potentiate the enzymatic degradation of the polysaccharides and their application might contribute to diminishing the currently prohibitive costs of the lignocellulose saccharification process.