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Cellohexaose

Cellohexaose O-CHE
Product code: O-CHE
€149.00

10 mg

Prices exclude VAT

Available for shipping

Content: 10 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 2478-35-5
Molecular Formula: C36H62O31
Molecular Weight: 990.9
Purity: > 95%
Substrate For (Enzyme): endo-Cellulase

High purity Cellohexaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

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Publications
Megazyme publication

Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.

Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.

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.

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Identification of a unique 1, 4-β-D-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.

Jiang, N., Ma, X. D., Fu, L. H., Li, C. X., Feng, J. X. & Duan, C. J. (2020). Applied Microbiology and Biotechnology, 104(16), 7051-7066.

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 kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km 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.

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Configuration of active site segments in lytic polysaccharide monooxygenases steers oxidative xyloglucan degradation.

Sun, P., Laurent, C. V., Scheiblbrandner, S., Frommhagen, M., Kouzounis, D., Sanders, M. G., van Berkel, W. J. H., Ludwig, R. & Kabel, M. A. (2020). Biotechnology for Biofuels, 13, 1-19.

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.

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Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field.

Stepnov, A. A., Fredriksen, L., Steen, I. H., Stokke, R. & Eijsink, V. G. (2019). PloS One, 14(9), e0222216.

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.

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A lytic polysaccharide monooxygenase from Myceliophthora thermophila and its synergism with cellobiohydrolases in cellulose hydrolysis.

Zhou, H., Li, T., Yu, Z., Ju, J., Zhang, H., Tan, H., Li, K. & Yin, H. (2019). International Journal of Biological Macromolecules, 139, 570-576.

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.

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Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.

Moreau, C., Tapin-Lingua, S., Grisel, S., Gimbert, I., Le Gall, S., Meyer, V., Petit-Conil, M., Berrin, J, Cathala, B. & Villares, A. (2019). Biotechnology for Biofuels, 12(1), 1-13.

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.

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An actinobacteria lytic polysaccharide monooxygenase acts on both cellulose and xylan to boost biomass saccharification.

Corrêa, T. L. R., Júnior, A. T., Wolf, L. D., Buckeridge, M. S., dos Santos, L. V. & Murakami, M. T. (2019). Biotechnology for Biofuels, 12(1), 117.

Background: Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. Results: The enzyme KpLPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, KpLPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of KpLPMO10A with Celluclast ® and an endo-β-1,4-xylanase also supports this finding. The crystallographic elucidation of KpLPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident diference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. Conclusions: This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic efect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass.

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A novel thermostable GH10 xylanase with activities on a wide variety of cellulosic substrates from a xylanolytic Bacillus strain exhibiting significant synergy with commercial Celluclast 1.5 L in pretreated corn stover hydrolysis.

Wang, K., Cao, R., Wang, M., Lin, Q., Zhan, R., Xu, H. & Wang, S. (2019). Biotechnology for Biofuels, 12(1), 48.

Background: Cellulose and hemicellulose are the two largest components in lignocellulosic biomass. Enzymes with activities towards cellulose and xylan have attracted great interest in the bioconversion of lignocellulosic biomass, since they have potential in improving the hydrolytic performance and reducing the enzyme costs. Exploring glycoside hydrolases (GHs) with good thermostability and activities on xylan and cellulose would be beneficial to the industrial production of biofuels and bio-based chemicals. Results: A novel GH10 enzyme (XynA) identified from a xylanolytic strain Bacillus sp. KW1 was cloned and expressed. Its optimal pH and temperature were determined to be pH 6.0 and 65°C. Stability analyses revealed that XynA was stable over a broad pH range (pH 6.0-11.0) after being incubated at 25°C for 24 h. Moreover, XynA retained over 95% activity after heat treatment at 60°C for 60 h, and its half-lives at 65°C and 70°C were about 12 h and 1.5 h, respectively. More importantly, in terms of substrate specificity, XynA exhibits hydrolytic activities towards xylans, microcrystalline cellulose (filter paper and Avicel), carboxymethyl cellulose (CMC), cellobiose, p-nitrophenyl-β-D-cellobioside (pNPC), and p-nitrophenyl-β-D-glucopyranoside (pNPG). Furthermore, the addition of XynA into commercial cellulase in the hydrolysis of pretreated corn stover resulted in remarkable increases (the relative increases may up to 90%) in the release of reducing sugars. Finally, it is worth mentioning that XynA only shows high amino acid sequence identity (88%) with rXynAHJ14, a GH10 xylanase with no activity on CMC. The similarities with other characterized GH10 enzymes, including xylanases and bifunctional xylanase/cellulase enzymes, are no more than 30%. Conclusions: XynA is a novel thermostable GH10 xylanase with a wide substrate spectrum. It displays good stability in a broad range of pH and high temperatures, and exhibits activities towards xylans and a wide variety of cellulosic substrates, which are not found in other GH10 enzymes. The enzyme also has high capacity in saccharification of pretreated corn stover. These characteristics make XynA a good candidate not only for assisting cellulase in lignocellulosic biomass hydrolysis, but also for the research on structure-function relationship of bifunctional xylanase/cellulase.

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Thermodynamic signatures of substrate binding for three Thermobifida fusca cellulases with different modes of action.

Hamre, A. G., Kaupang, A., Payne, C. M., Väljamäe, P. & Sørlie, M. (2019). Biochemistry, 58(12), 1648-1659.

The enzymatic breakdown of recalcitrant polysaccharides is achieved by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive, meaning that they stay bound to the substrate between subsequent catalytic interactions. Cellulases are GHs that catalyze the hydrolysis of cellulose [β-1,4-linked glucose (Glc)]. Here, we have determined the relative subsite binding affinity for a glucose moiety as well as the thermodynamic signatures for (Glc)6 binding to three of the seven cellulases produced by the bacterium Thermobifida fusca. TfCel48A is exo-processive, TfCel9A endo-processive, and TfCel5A endo-nonprocessive. Initial hydrolysis of (Glc)5 and (Glc)6 was performed in H218O enabling the incorporation of an 18O atom at the new reducing end anomeric carbon. A matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of the products reveals the intensity ratios of otherwise identical 18O- and 16O-containing products to provide insight into how the substrate is placed during productive binding. The two processive cellulases have significant binding affinity in subsites where products dissociate during processive hydrolysis, aligned with a need to have a pushing potential to remove obstacles on the substrate. Moreover, we observed a correlation between processive ability and favorable binding free energy, as previously postulated. Upon ligand binding, the largest contribution to the binding free energy is desolvation for all three cellulases as determined by isothermal titration calorimetry. The two endo-active cellulases show a more favorable solvation entropy change compared to the exo-active cellulase, while the two processive cellulases have less favorable changes in binding enthalpy compared to the nonprocessive TfCel5A.

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Functional characterization and comparative analysis of two heterologous endoglucanases from diverging subfamilies of glycosyl hydrolase family 45.

Berto, G. L., Velasco, J., Ribeiro, C. T. C., Zanphorlin, L. M., Domingues, M. N., Murakami, M. T., Polikarpoy, I., Oliveira, L. C., Ferraz, A. & Segato, F. (2019). Enzyme and Microbial Technology, 120, 23-35.

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.

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A novel thermostable GH3 β-glucosidase from Talaromyce leycettanus with broad substrate specificity and significant soybean isoflavone glycosides-hydrolyzing capability.

Reichenbach, T., Li, X., Xia, W., Bai, Y., Ma, R., Yang, H., Luo, H. & Shi, P. (2018). BioMed Research International, 2018, 4794690.

A novel β-glucosidase gene (Bgl3B) of glycoside hydrolase (GH) family 3 was cloned from the thermophilic fungus Talaromyce leycettanus JM12802 and successfully expressed in Pichia pastoris. The deduced Bgl3B contains 860 amino acid residues with a calculated molecular mass of 91.2 kDa. The purified recombinant Bgl3B exhibited maximum activities at pH 4.5 and 65°C and remained stable at temperatures up to 60°C and pH 3.0-9.0, respectively. The enzyme exhibited broad substrate specificities, showing β-glucosidase, glucanase, cellobiase, xylanase, and isoflavone glycoside hydrolase activities, and its activities were stimulated by short-chain alcohols. The catalytic efficiencies of Bgl3B were 693 and 104/mM/s towards pNPG and cellobiose, respectively. Moreover, Bgl3B was highly effective in converting isoflavone glycosides to aglycones at 37°C within 10 min, with the hydrolysis rates of 95.1%, 76.0%, and 75.3% for daidzin, genistin, and glycitin, respectively. These superior properties make Bgl3B potential for applications in the food, animal feed, and biofuel industries.

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Functional characterization of a lytic polysaccharide monooxygenase from the thermophilic fungus Myceliophthora thermophila.

Kadowaki, M. A., Várnai, A., Jameson, J. K., Leite, A. E., Costa-Filho, A. J., Kumagai, P. S., Prade, R. A., Polikarpov, I. & Eijsink, V. G. (2018). PloS One, 13(8), e0202148.

Thermophilic fungi are a promising source of thermostable enzymes able to hydrolytically or oxidatively degrade plant cell wall components. Among these enzymes are lytic polysaccharide monooxygenases (LPMOs), enzymes capable of enhancing biomass hydrolysis through an oxidative mechanism. Myceliophthora thermophila (synonym Sporotrichum thermophile), an Ascomycete fungus, expresses and secretes over a dozen different LPMOs. In this study, we report the overexpression and biochemical study of a previously uncharacterized LPMO (MtLPMO9J) from M. thermophila M77 in Aspergillus nidulansMtLPMO9J is a single-domain LPMO and has 63% sequence similarity with the catalytic domain of NcLPMO9C from Neurospora crassa. Biochemical characterization of MtLPMO9J revealed that it performs C4-oxidation and is active against cellulose, soluble cello-oligosaccharides and xyloglucan. Moreover, biophysical studies showed that MtLPMO9J is structurally stable at pH above 5 and at temperatures up to 50°C. Importantly, LC-MS analysis of the peptides after tryptic digestion of the recombinantly produced protein revealed not only the correct processing of the signal peptide and methylation of the N-terminal histidine, but also partial autoxidation of the catalytic center. This shows that redox conditions need to be controlled, not only during LPMO reactions but also during protein production, to protect LPMOs from oxidative damage.

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A highly glucose-tolerant GH1 β-glucosidase with greater conversion rate of soybean isoflavones in monogastric animals.

Cao, H., Zhang, Y., Shi, P., Ma, R., Yang, H., Xia, W., Cui, Y., Luo, H., Bai, Y. & Yao, B. (2018). Journal of Industrial Microbiology & Biotechnology, 197, 1-10.

In the feed industry, β-glucosidase has been widely used in the conversion of inactive and bounded soybean isoflavones into active aglycones. However, the conversion is frequently inhibited by the high concentration of intestinal glucose in monogastric animals. In this study, a GH1 β-glucosidase (AsBG1) with high specific activity, thermostability and glucose tolerance (IC50 = 800 mM) was identified. It showed great glucose tolerance against substrates with hydrophobic aryl ligands (such as pNPG and soy isoflavones). Using soybean meal as the substrate, AsBG1 exhibited higher hydrolysis efficiency than the GH3 counterpart Bgl3A with or without the presence of glucose in the reaction system. Furthermore, it is the first time to find that the endogenous β-glucosidase of soybean meal, mostly belonging to GH3, plays a role in the hydrolysis of soybean isoflavones and is highly sensitive to glucose. These findings lead to a conclusion that the GH1 rather than GH3 β-glucosidase has prosperous application advantages in the conversion of soybean isoflavones in the feed industry.

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Determination of optimal biomass pretreatment strategies for biofuel production: investigation of relationships between surface-exposed polysaccharides and their enzymatic conversion using carbohydrate-binding modules. 

Khatri, V., Meddeb-Mouelhi, F., Adjallé, K., Barnabé, S. & Beauregard, M. (2018). Biotechnology for Biofuels, 11(1), 144.

Background: Pretreatment of lignocellulosic biomass (LCB) is a key step for its efficient bioconversion into ethanol. Determining the best pretreatment and its parameters requires monitoring its impacts on the biomass material. Here, we used fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay to study the relationship between surface-exposed polysaccharides and enzymatic hydrolysis of LCB. Results: Our results indicated that alkali extrusion pretreatment led to the highest hydrolysis rates for alfalfa stover, cattail stems and flax shives, despite its lower lignin removal efficiency compared to alkali pretreatment. Corn crop residues were more sensitive to alkali pretreatments, leading to higher hydrolysis rates. A clear relationship was consistently observed between total surface-exposed cellulose detected by the FTCM-depletion assay and biomass enzymatic hydrolysis. Comparison of bioconversion yield and total composition analysis (by NREL/TP-510-42618) of LCB prior to or after pretreatments did not show any close relationship. Lignin removal efficiency and total cellulose content (by NREL/TP-510-42618) led to an unreliable prediction of enzymatic polysaccharide hydrolysis. Conclusions: Fluorescent protein-tagged carbohydrate-binding modules method (FTCM)-depletion assay provided direct evidence that cellulose exposure is the key determinant of hydrolysis yield. The clear and robust relationships that were observed between the cellulose accessibility by FTCM probes and enzymatic hydrolysis rates change could be evolved into a powerful prediction tool that might help develop optimal biomass pretreatment strategies for biofuel production.

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Performance, egg quality, nutrient digestibility, and excreta microbiota shedding in laying hens fed corn-soybean-meal-wheat-based diets supplemented with xylanase.

Lei, X. J., Lee, K. Y., & Kim, I. H. (2018). Poultry science, 97(6), 2071-2077.

The aim of this study was to evaluate the effects of dietary levels of xylanase on production performance, egg quality, nutrient digestibility, and excreta microbiota shedding of laying hens in a 12-week trial. Two-hundred-forty Hy-Line brown laying hens (44 wk old) were distributed according to a randomized block experimental design into one of 4 dietary treatments with 10 replicates of 6 birds each. The 4 dietary treatments were corn-soybean-meal-wheat-based diets supplemented with 0, 225, 450, or 900 U/kg xylanase. Daily feed intake, egg production, egg weight, egg mass, feed conversion ratio, and damaged egg rate showed no significant response to increasing xylanase supplementation during any phase (P > 0.05). No significant responses were observed for apparent total tract digestibility of dry matter, nitrogen, or gross energy (P > 0.05). A significant linear increase to increasing xylanase supplementation was seen for lactic acid bacteria numbers, although coliforms and Salmonella counts were not affected. Increasing the dietary xylanase resulted in a significant linear increase in eggshell thickness in wk 3, 6, 9, and 12 (P < 0.05). In addition, a significant linear increase occurred for Haugh unit and albumen height in wk 12 (P < 0.05). In summary, the inclusion of xylanase in corn-soybean-meal-wheat-based diets increased eggshell thickness, Haugh unit, albumen height, and excreta lactic acid bacteria count but had no effect on production performance or nutrient digestibility.

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A biorefinery approach for fractionation of Miscanthus lignocellulose using subcritical water extraction and a modified organosolv process.

Kubota, A. M., Kalnins, R. & Overton, T. W. (2018). Biomass and Bioenergy, 111, 52-59.

Using a biorefinery approach, biomass polymers such as lignin and carbohydrates can be selectively purified from lignocellulosic feedstocks with the aim of generating not only lignocellulosic bioethanol but also high value bio-based compounds. Furthermore, the efficient use of the entire biomass can increase overall feedstock value and significantly contribute to process cost-effectiveness. Therefore, the aim of this work was to fractionate the main compounds of the energy crop Miscanthus x giganteus (MxG) using ‘green’ solvents in order to obtain cellulose-enriched fibres as well as non-toxic streams rich in hemicellulose and lignin. Two processing routes were compared: a direct 1-step modified organosolv method for simultaneous lignin and hemicellulose removal; and a 3-step sequential process using subcritical water extraction for recovery of first extractives then hemicellulose, followed by modified organosolv lignin extraction. Both methods successfully generated cellulose-enriched fibres; from a complex mixture of compounds present in MxG, it was possible to obtain fibres comprising 78% cellulose without the use of commonly-applied toxic solvents that can potentially limit end uses for processed biomass and/or need additional neutralization steps. Fibres generated by the direct and sequential processes were very similar in composition; however, physicochemical analysis of the fibres using scanning electron microscopy, Fourier-transform infrared spectroscopy and principal component analysis confirmed structural differences resulting from the two processing routes, which were demonstrated to have an impact on downstream processing.

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Expression and characterization of the processive exo-β-1, 4-cellobiohydrolase SCO6546 from Streptomyces coelicolor A (3).

Lee, C. R., Chi, W. J., Lim, J. H., Dhakshnamoorthy, V. & Hong, S. K. (2018). Journal of basic microbiology, 58(4), 310-321.

The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and β-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50°C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM, for cellotetraose at pH 5.0 and 50°C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-β-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.

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Application of carbohydrate arrays coupled with mass spectrometry to detect activity of plant-polysaccharide degradative enzymes from the fungus Aspergillus niger.

van Munster, J. M., Thomas, B., Riese, M., Davis, A. L., Gray, C. J., Archer, D. B. & Flitsch, S. L. (2017). Scientific Reports, 7.

Renewables-based biotechnology depends on enzymes to degrade plant lignocellulose to simple sugars that are converted to fuels or high-value products. Identification and characterization of such lignocellulose degradative enzymes could be fast-tracked by availability of an enzyme activity measurement method that is fast, label-free, uses minimal resources and allows direct identification of generated products. We developed such a method by applying carbohydrate arrays coupled with MALDI-ToF mass spectrometry to identify reaction products of carbohydrate active enzymes (CAZymes) of the filamentous fungus Aspergillus niger. We describe the production and characterization of plant polysaccharide-derived oligosaccharides and their attachment to hydrophobic self-assembling monolayers on a gold target. We verify effectiveness of this array for detecting exo- and endo-acting glycoside hydrolase activity using commercial enzymes, and demonstrate how this platform is suitable for detection of enzyme activity in relevant biological samples, the culture filtrate of A. niger grown on wheat straw. In conclusion, this versatile method is broadly applicable in screening and characterisation of activity of CAZymes, such as fungal enzymes for plant lignocellulose degradation with relevance to biotechnological applications as biofuel production, the food and animal feed industry.

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RP-UHPLC-UV-ESI-MS/MS analysis of LPMO generated C4-oxidized gluco-oligosaccharides after non-reductive labeling with 2-aminobenzamide.

Frommhagen, M., van Erven, G., Sanders, M., van Berkel, W. J., Kabel, M. A. & Gruppen, H. (2017). Carbohydrate Research, 448, 191-199.

Lytic polysaccharide monooxygenases (LPMOs) are able to cleave recalcitrant polysaccharides, such as cellulose, by oxidizing the C1 and/or C4 atoms. The analysis of the resulting products requires a variety of analytical techniques. Up to now, these techniques mainly focused on the identification of non-oxidized and C1-oxidized oligosaccharides. The analysis of C4-oxidized gluco-oligosaccharides is mostly performed by using high pressure anion exchange chromatography (HPAEC). However, the alkaline conditions used during HPAEC analysis lead to tautomerization of C4-oxidized gluco-oligosaccharides, which limits the use of this technique. Here, we describe the use of reverse phase-ultra high performance liquid chromatography (RP-UHPLC) in combination with non-reductive 2-aminobenzamide (2-AB) labeling. Non-reductive 2-AB labeling enabled separation of C4-oxidized gluco-oligosaccharides from their non-oxidized counterparts. Moreover, RP-UHPLC does not require buffered mobile phases, which reduce mass spectrometry (MS) sensitivity. The latter is seen as an advantage over other techniques such as hydrophilic interaction liquid chromatography and porous graphitized carbon coupled to MS. RP-UHPLC coupled to UV detection and mass spectrometry allowed the identification of both labeled non-oxidized and C4-oxidized oligosaccharides. Non-reductive labeling kept the ketone at the C4-position of LPMO oxidized oligosaccharides intact, while selective reducing agents such as sodium triacetoxyborohydride (STAB) reduced this ketone group. Our results show that RP-UHPLC-UV-ESI-MS in combination with non-reductively 2-AB labeling is a suitable technique for the separation and identification of LPMO-generated C4-oxidized gluco-oligosaccharides.

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Recombinant expression of thermostable processive MtEG5 endoglucanase and its synergism with MtLPMO from Myceliophthora thermophila during the hydrolysis of lignocellulosic substrates.

Karnaouri, A., Muraleedharan, M. N., Dimarogona, M., Topakas, E., Rova, U., Sandgren, M. & Christakopoulos, P. (2017). Biotechnology for Biofuels, 10(1), 126.

Background: Filamentous fungi are among the most powerful cellulolytic organisms in terrestrial ecosystems. To perform the degradation of lignocellulosic substrates, these microorganisms employ both hydrolytic and oxidative mechanisms that involve the secretion and synergism of a wide variety of enzymes. Interactions between these enzymes occur on the level of saccharification, i.e., the release of neutral and oxidized products, but sometimes also reflected in the substrate liquefaction. Although the synergism regarding the yield of neutral sugars has been extensively studied, further studies should focus on the oxidized sugars, as well as the effect of enzyme combinations on the viscosity properties of the substrates. Results: In the present study, the heterologous expression of an endoglucanase (EG) and its combined activity together with a lytic polysaccharide monooxygenase (LPMO), both from the thermophilic fungus Myceliophthora thermophila, are described. The EG gene, belonging to the glycoside hydrolase family 5, was functionally expressed in the methylotrophic yeast Pichia pastoris. The produced Mt EG5A (75 kDa) featured remarkable thermal stability and showed high specific activity on microcrystalline cellulose compared to CMC, which is indicative of its processivity properties. The enzyme was capable of releasing high amounts of cellobiose from wheat straw, birch, and spruce biomass. Addition of MtLPMO9 together with MtEG5A showed enhanced enzymatic hydrolysis yields against regenerated amorphous cellulose (PASC) by improving the release not only of the neutral but also of the oxidized sugars. Assessment of activity of MtEG5A on the reduction of viscosity of PASC and pretreated wheat straw using dynamic viscosity measurements revealed that the enzyme is able to perform liquefaction of the model substrate and the natural lignocellulosic material, while when added together with MtLPMO9, no further synergistic effect was observed. Conclusions: The endoglucanase MtEG5A from the thermophilic fungus M. thermophila exhibited excellent properties that render it a suitable candidate for use in biotechnological applications. Its strong synergism with LPMO was reflected in sugars release, but not in substrate viscosity reduction. Based on the level of oxidative sugar formation, this is the first indication of synergy between LPMO and EG reported.

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Characterization of a novel theme C glycoside hydrolase family 9 cellulase and its CBM-chimeric enzymes.

Duan, C. J., Huang, M. Y., Pang, H., Zhao, J., Wu, C. X. & Feng, J. X. (2017). Applied Microbiology and Biotechnology, 1-15.

In bacterial cellulase systems, glycoside hydrolase family 9 (GH9) cellulases are generally regarded as the major cellulose-degrading factors besides GH48 exoglucanase. In this study, umcel9A, which was cloned from uncultured microorganisms from compost, with the encoded protein being theme C GH9 cellulase, was heterologously expressed in Escherichia coli, and the biochemical properties of the purified enzyme were characterized. Hydrolysis of carboxylmethylcellulose (CMC) by Umcel9A led to the decreased viscosity of CMC solution and production of reducing sugars. Interestingly, cellobiose was the major product when cellulosic materials were hydrolyzed by Umcel9A. Six representative carbohydrate-binding modules (CBMs) from different CBM families (CBM1, CBM2, CBM3, CBM4, CBM10, and CBM72) were fused with Umcel9A at the natural terminal position, resulting in significant enhancement of the binding capacity of the chimeric enzymes toward four different insoluble celluloses as compared with that of Umcel9A. Catalytic activity of the chimeric enzymes against insoluble celluloses, including phosphoric acid-swollen cellulose (PASC), alkali-pretreated sugarcane bagasse (ASB), filter paper powder (FPP), and Avicel, was higher than that of Umcel9A, except for Umcel9A-CBM3. In these chimeric enzymes, CBM4-Umcel9A exhibited the highest activity toward the four tested insoluble celluloses and displayed 4.2-, 3.0-, 2.4-, and 6.6-fold enhanced activity toward PASC, ASB, FPP, and Avicel, respectively, when compared with that of Umcel9A. CBM4-Umcel9A also showed highest Vmax and catalytic efficiency (kcat/KM) against PASC. Construction of chimeric enzymes may have potential applications in biocatalytic processes and provides insight into the evolution of the molecular architecture of catalytic module and CBM in GH9 cellulases.

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Novel archaeal thermostable cellulases from an oil reservoir metagenome.

Lewin, A., Zhou, J., Pham, V. T. T., Haugen, T., El Zeiny, M., Aarstad, O., Aarstad, O., Liebl, W., Wentzel, A. & Liles, M. R. (2017). AMB Express, 7(1), 183.

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.

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Biochemical studies of two lytic polysaccharide monooxygenases from the white-rot fungus Heterobasidion irregulare and their roles in lignocellulose degradation.

Liu, B., Olson, Å., Wu, M., Broberg, A. & Sandgren, M. (2017). PloS One, 12(12), e0189479.

Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris. The oxidization preferences and substrate specificities of these two enzymes were determined. The two LPMOs were shown to cleave different carbohydrate components of plant cell walls. HiLPMO9H was active on cellulose and oxidized the substrate at the C1 carbon of the pyranose ring at β-1,4-glycosidic linkages, whereas HiLPMO9I cleaved cellulose with strict oxidization at the C4 carbon of glucose unit at internal bonds, and also showed activity against glucomannan. We propose that the two LPMOs play different roles in the plant-cell-wall degrading system of H. irregulare for degradation of softwood and that the lignocellulose degradation mediated by this white-rot fungus may require collective efforts from multi-types of LPMOs.

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A thermostable Gloeophyllum trabeum xylanase with potential for the brewing industry.

Wang, X., Luo, H., Yu, W., Ma, R., You, S., Liu, W., Hou, L., Zheng, F., Xie, X. & Yao, B. (2016). Food Chemistry, 199, 516-523.

A xylanase gene of glycoside hydrolase family 10, GtXyn10, was cloned from Gloeophyllum trabeum CBS 900.73 and expressed in Pichia pastoris GS115. Purified recombinant GtXyn10 exhibited significant activities to xylan (100.0%), lichenan (11.2%), glucan (15.2%) and p-nitrophenol-β-cellobiose (18.6%), demonstrated the maximum xylanase and glucanase activities at pH 4.5–5.0 and 75°C, retained stability over the pH range of 2.0–7.5 and at 70°C, and was resistant to pepsin and trypsin, most metal ions and SDS. Multiple sequence alignment and modeled-structure analysis identified a unique Gly48 in GtXyn10, and site-directed mutagenesis of Gly48 to Lys improved the temperature optimum up to 80°C. Under simulated mashing conditions, GtXyn10 (80 U) reduced the mash viscosity by 12.8% and improved the filtration rate by 31.3%. All these properties above make GtXyn10 attractive for potential applications in the feed and brewing industries.

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Identification and characterization of plant cell wall degrading enzymes from three glycoside hydrolase families in the cerambycid beetle Apriona japonica.

Pauchet, Y., Kirsch, R., Giraud, S., Vogel, H. & Heckel, D. G. (2014). Insect Biochemistry and Molecular Biology, 49, 1-13.

Xylophagous insects have evolved to thrive in a highly challenging environment. For example, wood-boring beetles from the family Cerambycidae feed exclusively on woody tissues, and to efficiently access the nutrients present in this sub-optimal environment, they have to cope with the lignocellulose barrier. Whereas microbes of the insect's gut flora were hypothesized to be responsible for the degradation of lignin, the beetle itself depends heavily on the secretion of a range of enzymes, known as plant cell wall degrading enzymes (PCWDEs), to efficiently digest both hemicellulose and cellulose networks. Here we sequenced the larval gut transcriptome of the Mulberry longhorn beetle, Apriona japonica (Cerambycidae, Lamiinae), in order to investigate the arsenal of putative PCWDEs secreted by this species. We combined our transcriptome with all available sequencing data derived from other cerambycid beetles in order to analyze and get insight into the evolutionary history of the corresponding gene families. Finally, we heterologously expressed and functionally characterized the A. japonica PCWDEs we identified from the transcriptome. Together with a range of endo-β-1,4-glucanases, we describe here for the first time the presence in a species of Cerambycidae of (i) a xylanase member of the subfamily 2 of glycoside hydrolase family 5 (GH5 subfamily 2), as well as (ii) an exopolygalacturonase from family GH28. Our analyses greatly contribute to a better understanding of the digestion physiology of this important group of insects, many of which are major pests of forestry worldwide.

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Isolation and identification of phenolic glucosides from thermally treated olive oil byproducts.

Rubio-Senent, F., Lama-Muñoz, A., Rodríguez-Gutiérrez, G. & Fernández-Bolaños, J. (2013). Journal of Agricultural and Food Chemistry, 61(6), 1235-1248.

A liquid phase rich in bioactive compounds, such as phenols and sugars, is obtained from olive oil waste by novel thermal treatment. Two groups of fractions with common characteristics were obtained and studied after thermal treatment, acid hydrolysis, and separation by ultrafiltration, chromatography, and finally Superdex Peptide HR. In the first group, which eluted at the same time as oligosaccharides with a low DP (4–2), an oleosidic secoiridoid structure conjugated to a phenolic compound (hydroxytyrosol) was identified as oleuropeinic acid, and three possible structures were detected. In the second group, glucosyl structures formed by hydroxytyrosol and one, two, or three units of glucose or by tyrosol and glucose have been proposed. Verbascoside, a heterosidic ester of caffeic acid, in which hydroxytyrosol is linked to rhamnose–glucose or one of its isomers was also identified. Neutral oligosaccharides bound to a phenol-containing compound could be antioxidant-soluble fibers with bioactive properties.

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Structure and Function of a Novel Cellulase 5 from Sugarcane Soil Metagenome.

Alvarez, T. M., Paiva, J. H., Ruiz, D. M., Cairo, J. P. L. F., Pereira, I. O., Paixão, D. A. A., de Almeida, R. F., Tonoli, C. C. C., Ruller, R., Santos, C. R., Squina, F. M. & Murakami, M. T. (2013). PloS one, 8(12), e83635.

Cellulases play a key role in enzymatic routes for degradation of plant cell-wall polysaccharides into simple and economically-relevant sugars. However, their low performance on complex substrates and reduced stability under industrial conditions remain the main obstacle for the large-scale production of cellulose-derived products and biofuels. Thus, in this study a novel cellulase with unusual catalytic properties from sugarcane soil metagenome (CelE1) was isolated and characterized. The polypeptide deduced from the celE1 gene encodes a unique glycoside hydrolase domain belonging to GH5 family. The recombinant enzyme was active on both carboxymethyl cellulose and β-glucan with an endo-acting mode according to capillary electrophoretic analysis of cleavage products. CelE1 showed optimum hydrolytic activity at pH 7.0 and 50°C with remarkable activity at alkaline conditions that is attractive for industrial applications in which conventional acidic cellulases are not suitable. Moreover, its three-dimensional structure was determined at 1.8 Å resolution that allowed the identification of an insertion of eight residues in the β8-α8 loop of the catalytic domain of CelE1, which is not conserved in its psychrophilic orthologs. This 8-residue-long segment is a prominent and distinguishing feature of thermotolerant cellulases 5 suggesting that it might be involved with thermal stability. Based on its unconventional characteristics, CelE1 could be potentially employed in biotechnological processes that require thermotolerant and alkaline cellulases.

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Kinetics of the enzymatic cellulose hydrolysis by the endoglucanase from the extremophile S. solfataricus.

Bonhage, B., Seiferheld, B. & Spiess, A. C. (2013). In Kraslawski A, Turunen I (eds.). Proceedings of the 23rd European Symposium on Computer Aided Process Engineering, 23, 85-90.

The hydrolysis of cellulose is a necessary step to provide sugars from biomass, e.g. for fermentation. A promising approach is to hydrolyse cellulose enzymatically. Naturally, cellulolytic enzymes appear in mixtures of at least four different enzyme activities. Until now, research has focused on these enzyme mixtures. But, to accurately describe cellulose hydrolysis, it is essential to identify the individual kinetic parameters of the employed cellulases. Therefore, we investigated the behaviour of the extremophile endoglucanase (EG) SSO1354 from S. solfataricus on cello-oligomers (COs) to determine its kinetic performance. The properties of interest were the binding affinity as function of the chain length of the cellulose as well as inhibitory and activating effects of short COs. We monitored the evolution of the chain length distribution over the reaction time using thin layer chromatography and the formation of reducing sugars with a colorimetric assay. According to the measurements, the cellulase requires a chain length of four or more glucose units to be catalytically active and the enzyme gets more active with increasing chain length. Also, cellotriose (C3) is an inhibitor for the used EG, and cellobiose (C2) seems to be an enzyme activator, in contrast to literature. With the obtained results it should be possible to mechanistically describe the hydrolysis of cellulose.

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Studies of enzymatic cleavage of cellulose using polysaccharide analysis by carbohydrate gel electrophoresis (PACE).

Kosik, O., Bromley, J. R., Busse-Wicher, M., Zhang, Z. & Dupree, P. (2012). Methods in Enzymology, 510, 51-67.

With the advent of fast genome analysis, many genes encoding novel putative cellulolytic enzymes are being identified in diverse bacterial and fungal genomes. The discovery of these genes calls for quick, robust, and reliable methods for qualitative and quantitative characterization of the enzymatic activities of the encoded proteins. Here, we describe the use of the polysaccharide analysis by carbohydrate gel electrophoresis (PACE) method, which was previously used, among other applications, to characterize various hemicellulose degrading enzymes; for structural elucidation of these carbohydrates; and for analysis of products resulting from enzymatic cleavage of cellulose. PACE relies on fluorescent labeling of mono-, oligo-, and polysaccharides at their reducing end and separation of the labeled carbohydrates by polyacrylamide gel electrophoresis. Labeling can be carried out before or after enzymatic digestion. PACE is very sensitive and allows analysis of both substrate specificities and kinetic properties of cellulolytic enzymes.

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A novel exo-cellulase from white spotted longhorn beetle (Anoplophora malasiaca).

Chang, C. J., Wu, C. P., Lu, S. C., Chao, A. L., Ho, T. H. D., Yu, S. M. & Chao, Y. C. (2012). Insect Biochemistry and Molecular Biology, 42(9), 629-636.

Wood feeding insects depends heavily on the secretion of a combination of cellulases, mainly endoglucanases and other glucanases such as exoglucanases and xylanases, for efficient digestion of the cellulosic materials. To date, although a high number of endoglucanases have been found in xytophagous insects, little is known about exoglucanases encoded in the genome of these insects. Here we report the identification and isolation of an exoglucanase, designated as AmCel-5B, from the white spotted longhorn beetle, Anoplophora malasiaca. The optimal condition of enzymatic activity was found to be 50°C and pH 4.0. Interestingly, this enzyme is not only exhibited exo-β-glucanase activity, but also with obvious endo-β-glucanase activity. Furthermore, this enzyme is unique in that, although it recognizes Avicel, evidenced as an exo-β-glucanase, it cannot recognize oligosaccharides smaller than cellohexaose. This may explain why longhorn beetle can well digest hard “living” wood, which contains primarily rigid long fibers. Although it is known that metal ions can enhance the activity of some cellulases, we further demonstrated that reducing agent could work synergistically with metal ions for significant activity enhancement of AmCel-5B. The discovery and investigation of an insect exoglucanase should lead to a greater understanding of the mechanism for efficient digestion of cellulosic materials by wood feeding insects, as well as facilitate their potential applications in the production of bioenergy and biomaterials from lignocellulosic biomass in the future.

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β-Glucosidases from a new Aspergillus species can substitute commercial β-glucosidases for saccharification of lignocellulosic biomass.

Sørensen, A., Lübeck, P. S., Lübeck, M., Teller, P. J. & Ahring, B. K. (2011). Canadian Journal of Microbiology, 57(8), 638-650.

β-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for β-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus) showed 10 times greater β-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial β-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis–Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4°C for Novozym 188. The significant thermostability of strain AP β-glucosidases was further confirmed when compared with Cellic CTec. The β-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for β-glucosidase activity.

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Functional diversity of four glycoside hydrolase family 3 enzymes from the rumen bacterium Prevotella bryantii B14.

Dodd, D., Kiyonari, S., Mackie, R. I. & Cann, I. K. O. (2010). Journal of Bacteriology, 192(9), 2335-2345.

Prevotella bryantii B14 is a member of the phylum Bacteroidetes and contributes to the degradation of hemicellulose in the rumen. The genome of P. bryantii harbors four genes predicted to encode glycoside hydrolase (GH) family 3 (GH3) enzymes. To evaluate whether these genes encode enzymes with redundant biological functions, each gene was cloned and expressed in Escherichia coli. Biochemical analysis of the recombinant proteins revealed that the enzymes exhibit different substrate specificities. One gene encoded a cellodextrinase (CdxA), and three genes encoded β-xylosidase enzymes (Xyl3A, Xyl3B, and Xyl3C) with different specificities for either para-nitrophenyl (pNP)-linked substrates or substituted xylooligosaccharides. To identify the amino acid residues that contribute to catalysis and substrate specificity within this family of enzymes, the roles of conserved residues (R177, K214, H215, M251, and D286) in Xyl3B were probed by site-directed mutagenesis. Each mutation led to a severely decreased catalytic efficiency without a change in the overall structure of the mutant enzymes. Through amino acid sequence alignments, an amino acid residue (E115) that, when mutated to aspartic acid, resulted in a 14-fold decrease in the Kcat/Km for pNP-β-D-xylopyranoside (pNPX) with a concurrent 1.1-fold increase in theKcat/Km for pNP-β-D-glucopyranoside (pNPG) was identified. Amino acid residue E115 may therefore contribute to the discrimination between β-xylosides and β-glucosides. Our results demonstrate that each of the four GH3 enzymes has evolved to perform a specific role in lignopolysaccharide hydrolysis and provide insight into the role of active-site residues in catalysis and substrate specificity for GH3 enzymes.

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The family 6 carbohydrate binding module CmCBM6-2 contains two ligand-binding sites with distinct specificities.

Henshaw, J. L., Bolam, D. N., Pires, V. M. R., Czjzek, M., Henrissat, B., Ferreira, L. M. A., Fontes. C. M. G. A. & Gilbert, H. J. (2004). Journal of Biological Chemistry, 279(20), 21552-21559.

The microbial degradation of the plant cell wall is an important biological process, representing a major component of the carbon cycle. Enzymes that mediate the hydrolysis of this composite structure are modular proteins that contain non-catalytic carbohydrate binding modules (CBMs) that enhance catalytic activity. CBMs are grouped into sequence-based families, and in a previous study we showed that a family 6 CBM (CBM6) that interacts with xylan contains two potential ligand binding clefts, designated cleft A and cleft B. Mutagenesis and NMR studies showed that only cleft A in this protein binds to xylan. Family 6 CBMs bind to a range of polysaccharides, and it was proposed that the variation in ligand specificity observed in these proteins reflects the specific cleft that interacts with the target carbohydrate. Here the biochemical properties of the C-terminal cellulose binding CBM6 (CmCBM6-2) from Cellvibrio mixtus endoglucanase 5A were investigated. The CBM binds to the β1,4-β1,3-mixed linked glucans lichenan and barley β-glucan, cello-oligosaccharides, insoluble forms of cellulose, the β1,3-glucan laminarin, and xylooligosaccharides. Mutagenesis studies, informed by the crystal structure of the protein (presented in the accompanying paper, Pires, V. M. R., Henshaw, J. L., Prates, J. A. M., Bolam, D., Ferreira, L. M. A. Fontes, C. M. G. A., Henrissat, B., Planas, A., Gilbert, H. J., Czjzek, M. (2004) J. Biol. Chem. 279, 21560-21568), show that both cleft A and B can accommodate cello-oligosaccharides and laminarin displays a preference for cleft A, whereas xylooligosaccharides exhibit absolute specificity for this site, and the β1,4,-β1,3-mixed linked glucans interact only with cleft B. The binding of CmCBM6-2 to insoluble cellulose involves synergistic interactions between cleft A and cleft B. These data show that CmCBM6-2 contains two binding sites that display differences in ligand specificity, supporting the view that distinct binding clefts with different specificities can contribute to the variation in ligand recognition displayed by family 6 CBMs. This is in sharp contrast to other CBM families, where variation in ligand binding is a result of changes in the topology of a single carbohydrate-binding site.

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Processivity, synergism, and substrate specificity of Thermobifida fusca Cel6B.

Vuong, T. V. & Wilson, D. B. (2009). Applied and Environmental Microbiology, 75(21), 6655-6661.

A relationship between processivity and synergism has not been reported for cellulases, although both characteristics are very important for hydrolysis of insoluble substrates. Mutation of two residues located in the active site tunnel of Thermobifida fusca exocellulase Cel6B increased processivity on filter paper. Surprisingly, mixtures of the Cel6B mutant enzymes and T. fusca endocellulase Cel5A did not show increased synergism or processivity, and the mutant enzyme which had the highest processivity gave the poorest synergism. This study suggests that improving exocellulase processivity might be not an effective strategy for producing improved cellulase mixtures for biomass conversion. The inverse relationship between the activities of many of the mutant enzymes with bacterial microcrystalline cellulose and their activities with carboxymethyl cellulose indicated that there are differences in the mechanisms of hydrolysis for these substrates, supporting the possibility of engineering Cel6B to target selected substrates.

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Clostridium thermocellum cellulase CelT, a family 9 endoglucanase without an Ig-like domain or family 3c carbohydrate-binding module.

Kurokawa, J., Hemjinda, E., Arai, T., Kimura, T., Sakka, K. & Ohmiya, K. (2002). Applied Microbiology and Biotechnology, 59(4), 455-461.

The celT gene of Clostridium thermocellum strain F1 was found downstream of the mannanase gene man26B [Kurokawa J et al. (2001) Biosci Biotechnol Biochem 65:548–554] in pKS305. The open reading frame of celT consists of 1,833 nucleotides encoding a protein of 611 amino acids with a predicted molecular weight of 68,510. The mature form of CelT consists of a family 9 cellulase domain and a dockerin domain responsible for cellulosome assembly, but lacks a family 3c carbohydrate-binding module (CBM) and an immunoglobulin (Ig)-like domain, which are often found with family 9 catalytic domains. CelT devoid of the dockerin domain (CelTΔdoc) was constructed and purified from a recombinant Escherichia coli, and its enzyme properties were examined. CelTΔdoc showed strong activity toward carboxymethylcellulose (CMC) and barley β-glucan, and low activity toward xylan. The Vmax and Km values were 137 µmol min-1 mg-1 and 16.7 mg/ml, respectively, for CMC. Immunological analysis indicated that CelT is a catalytic component of the C. thermocellum F1 cellulosome. This is the first report describing the characterization of a family 9 cellulase without an Ig-like domain or family 3c CBM.

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