Cellotetraose

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

An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC₅₀ = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases.

Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.

Banana production generates significant amounts of agricultural wastes, being fiber extraction one of the most relevant alternatives for their valorization. This process produces banana’s pseudostem pulp (BPP) as a byproduct, which shows an interesting composition for the biorefinery’s biochemical platform, with high polysaccharides (68%) and low lignin contents. This work deals with the enzymatic hydrolysis (EH) of raw and hydrothermally pre-treated BPP, focusing on the production of oligosaccharides (OS). Raw BPP hydrolysis with cellulase at different dosages rendered only 3.2% OS yields (OSY). Pectinase addition has not affected EH performance. On the other hand, EH of hydrothermally pre-treated BPP at 150°C and 170°C (P150 and P170) allowed to increase OSY up to 28% (P150, 1 FPU of cellulase/g dry biomass, 12 h), being 72% of the solubilized sugars in the form of cello-oligosaccharides. This last condition was subjected to a multi-stage EH strategy without improvements in OSY. An endo-glucanase was also tested, but obtained OSY were lower than cellulase results. Finally, obtained OS demonstrated to stimulate the growth of two Lactobacilli strains. The results show that BPP pre-treated under mild operational conditions is a good candidate for cello-oligosaccharides production by EH using 1 FPU/g DB of cellulase with a simple strategy.

Many phytopathogens secrete cell wall degradation enzymes (CWDEs) to damage host cells and facilitate colonization. As the major components of the plant cell wall, cellulose and hemicellulose are the targets of CWDEs. Damaged plant cells often release damage-associated molecular patterns (DAMPs) to trigger plant immune responses. Here, we establish that the fungal pathogen Magnaporthe oryzae secretes the endoglucanases MoCel12A and MoCel12B during infection of rice (Oryza sativa). These endoglucanases target hemicellulose of the rice cell wall and release two specific oligosaccharides, namely the trisaccharide 3¹-β-D-Cellobiosyl-glucose and the tetrasaccharide 3¹-β-D-Cellotriosyl-glucose. 3¹-β-D-Cellobiosyl-glucose and 3¹-β-D-Cellotriosyl-glucose bind the immune receptor OsCERK1 but not the chitin binding protein OsCEBiP. However, they induce the dimerization of OsCERK1 and OsCEBiP. In addition, these Poaceae cell wall-specific oligosaccharides trigger a burst of reactive oxygen species (ROS) that is largely compromised in oscerk1 and oscebip mutants. We conclude that 3¹-β-D-Cellobiosyl-glucose and 3¹-β-D-Cellotriosyl-glucose are specific DAMPs released from the hemicellulose of rice cell wall, which are perceived by an OsCERK1 and OsCEBiP immune complex during M. oryzae infection in rice.

Background: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.

Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley β-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50°C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (β/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability. Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product.

Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestrit (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.

A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.

Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-β-D-glucan glucohydrolase (EC 3.2.1.74) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl β-D-cellobioside (ρ-NPC) and ρ-nitrophenyl β-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The k_cat/K_m values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its k_cat/K_m for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.

This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200°C; 15 bar; 10 min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500 g/kg of initial xylan in the biomass after only 4 h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.

This paper addresses two fundamental issues in the peak deconvolution method of cellulose XRD data analysis: there is no standard model for amorphous cellulose and common peak functions such as Gauss, Lorentz and Voigt functions do not fit the amorphous profile well. It first examines the effects of ball milling on three types of cellulose and results show that ball milling transforms all samples into a highly amorphous phase exhibiting nearly identical powder X-ray diffraction (XRD) profiles. It is hypothesized that short range order within a glucose unit and between adjacent units survives ball milling and generates the characteristic amorphous XRD profiles. This agrees well with cellulose I d-spacing measurements and oligosaccharide XRD analysis. The amorphous XRD profile is modeled using a Fourier series equation where the coefficients are determined using the nonlinear least squares method. A new peak deconvolution method then is proposed to analyze cellulose XRD data with the amorphous Fourier model function in conjunction with standard Voigt functions representing the crystalline peaks. The impact of background subtraction method has also been assessed. Analysis of several cellulose samples was then performed and compared to the conventional peak deconvolution methods with common peak fitting functions and background subtraction approach. Results suggest that prior peak deconvolution methods overestimate cellulose crystallinity.

The notion that xyloglucans (XG) play a pivotal role in tethering cellulose microfibrils in the primary cell wall of plants can be traced back to the first molecular model of the cell wall proposed in 1973, which was reinforced in the 1990s by the identification of Xyloglucan Endotransglucosylase/Hydrolase (XTH) enzymes that cleave and reconnect xyloglucan crosslinks in the cell wall. However, this tethered network model has been seriously challenged since 2008 by the identification of the Arabidopsis thaliana xyloglucan-deficient mutant (xxt1 xxt2), which exhibits functional cell walls. Thus, the molecular mechanism underlying the physical integration of cellulose microfibrils into the cell wall remains controversial. To resolve this dilemma, we investigated the cell wall regeneration process using mesophyll protoplasts derived from xxt1 xxt2 mutant leaves. Imaging analysis revealed only a slight difference in the structure of cellulose microfibril network between xxt1 xxt2 and wild-type (WT) protoplasts. Additionally, exogenous xyloglucan application did not alter the cellulose deposition patterns or mechanical stability of xxt1 xxt2 mutant protoplasts. These results indicate that xyloglucan is not essential for the initial assembly of the cellulose network, and the cellulose network formed in the absence of xyloglucan provides sufficient tensile strength to the primary cell wall regenerated from protoplasts.

This study investigated pilot-scale production of xylo-oligosaccharides (XOS) and fermentable sugars from Miscanthus using steam explosion (SE) pretreatment. SE conditions (200°C; 15 bar; 10 min) led to XOS yields up to 52 % (w/w of initial xylan) in the hydrolysate. Liquid chromatography-mass spectrometry demonstrated that the solubilised XOS contained bound acetyl- and hydroxycinnamate residues, physicochemical properties known for high prebiotic effects and anti-oxidant activity in nutraceutical foods. Enzymatic hydrolysis of XOS-rich hydrolysate with commercial endo-xylanases resulted in xylobiose yields of 380 to 500 g/kg of initial xylan in the biomass after only 4 h, equivalent to ~74 to 90 % conversion of XOS into xylobiose. Fermentable glucose yields from enzymatic hydrolysis of solid residues were 8 to 9-fold higher than for untreated material. In view of an integrated biorefinery, we demonstrate the potential for efficient utilisation of Miscanthus for the production of renewable sources, including biochemicals and biofuels.

Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC). Results: The fungal LPMO from AA9 family (PaLPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with PaLPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC. Conclusions: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.

Lignocellulosic materials are abundant, renewable and are emerging as valuable substrates for many industrial applications such as the production of second-generation biofuels, green chemicals and pharmaceuticals. However, the recalcitrance and the complexity of cell wall polysaccharides require multiple enzymes for their complete conversion to oligo- and monosaccharides. The endoglucanases from GH45 family are a small and relatively poorly studied group of enzymes with potential industrial application. The present study reports cloning, heterologous expression and functional characterization of two GH45 endoglucanases from mesophilic fungi Gloeophyllum trabeum (GtGH45) and thermophilic fungi Myceliophthora thermophila (MtGH45), which belong to subfamilies GH45C and GH45A, respectively. Both enzymes have optimal pH 5.0 and melting temperatures (Tm) of 66.0°C and 80.9°C, respectively, as estimated from circular dichroism experiments. The recombinant proteins also exhibited different mode of action when incubated with oligosaccharides ranging from cellotriose to cellohexaose, generating mainly cellobiose and cellotriose (MtGH45) or glucose and cellobiose (GtGH45). The MtGH45 did not show activity against oligosaccharides smaller than cellopentaose while the enzyme GtGH45 was able to depolymerize cellotriose, however with lower efficiency when compared to larger oligosaccharides. Furthermore, both GHs45 were stable up to 70°C for 24 h and useful to enhance initial glucan hydrolysis rates during saccharification of sugarcane pith by a mixture of cellulolytic enzymes. Recombinant GHs45 from diverging subfamilies stand out for differences in substrate specificity appearing as new tools for preparation of enzyme cocktails used in cellulose hydrolysis.

Using multi-step error prone PCR (ep-PCR) of the gene encoding endoglucanase Cel12A (27 kDa) from Thermotoga neapolitana, mutants were obtained with many fold increase in the enzyme activity. The best mutant (C6, N47S/E57 K/ V88A/S157 P/K165 H) obtained after four rounds of ep-PCR showed 2.7−, 5− and 4.8−fold increase in activity against CMC, RAC and Avicel, respectively, compared with the wild type enzyme. The other characteristics of the mutated enzyme with respect to stability, optimum working pH and temperature were comparable to the wild type enzyme.C6 mutant showed higher binding efficiency towards the rice straw (∼50%) than the wild type (∼41%). The structural information obtained from the protein docking of the wild type Cel12A and its mutant showed that E57 K improved the binding affinity between enzyme and ligand by producing conformational changes in the catalytic cavity. The other mutations can facilitate the enzyme-substrate binding interactions to enhance catalytic activity although they are not directly involved in catalysis. The wild type and mutant enzyme produce cellobiose as the major products for both soluble and insoluble substrates, suggesting that this enzyme should be a cellobiohydrolase instead of endoglucanase as previously reported.

A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A.

Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.