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Cellotriose
Product code: O-CTR-50MG

Content:

€156.00

50 mg

Prices exclude VAT

Available for shipping

Content: 50 mg or 100 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient or Below -10oC
Physical Form: Powder
Stability: > 10 years under recommended storage conditions
CAS Number: 33404-34-1
Molecular Formula: C18H32O16
Molecular Weight: 504.4
Purity: > 95%
Substrate For (Enzyme): endo-Cellulase

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

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Data booklets for each pack size are located in the Documents tab.

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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Improved cellulose X-ray diffraction analysis using Fourier series modeling.

Yao, W., Weng, Y. & Catchmark, J. M. (2020).  Cellulose, 1-17.

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.

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Pilot-scale production of xylo-oligosaccharides and fermentable sugars from Miscanthus using steam explosion pretreatment.

Bhatia, R., Winters, A., Bryant, D. N., Bosch, M., Clifton-Brown, J., Leak, D. & Gallagher, J. (2020). Bioresource Technology, 296, 122285.

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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Improvement in activity of cellulase Cel12A of Thermotoga neapolitana by error prone PCR.

Basit, A., Tajwar, R., Sadaf, S., Zhang, Y. & Akhtar, M. W. (2019). Journal of Biotechnology, 306, 118-124.

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.

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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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Turning a potent family‐9 free cellulase into an operational cellulosomal component and vice versa.

Vita, N., Borne, R., Perret, S., de Philip, P. & Fierobe, H. P. (2019). The FEBS Journal, 286(17), 3359-3373.

Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family‐9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, whereas its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In this study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C‐terminal moiety of Cel9A encompassing the two X2 modules and the family‐3b carbohydrate binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family‐48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family‐9 cellulase.

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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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The carbohydrate-binding module and linker of a modular lytic polysaccharide monooxygenase promote localized cellulose oxidation.

Courtade, G., Forsberg, Z., Heggset, E. B., Eijsink, V. G. & Aachmann, F. L. (2018).  Journal of Biological Chemistry, 293(34), 13006-13015.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that catalyze the oxidative cleavage of polysaccharides such as cellulose and chitin, a feature that makes them key tools in industrial biomass conversion processes. The catalytic domains of a considerable fraction of LPMOs and other carbohydrate-active enzymes (CAZymes) are tethered to carbohydrate-binding modules (CBMs) by flexible linkers. These linkers preclude X-ray crystallographic studies, and the functional implications of these modular assemblies remain partly unknown. Here, have used NMR spectroscopy to characterize structural and dynamic features of full-length modular ScLPMO10C from Streptomyces coelicolor. We observed that the linker is disordered and extended, creating distance between the CBM and the catalytic domain and allowing these domains to move independently of each other. Functional studies with cellulose nanofibrils revealed that most of the substrate-binding affinity of full-length ScLPMO10C resides in the CBM. Comparison of the catalytic performance of full-length ScLPMO10C and its isolated catalytic domain revealed that the CBM is beneficial for LPMO activity at lower substrate concentrations and promotes localized and repeated oxidation of the substrate. Taken together, these results provide a mechanistic basis for understanding the interplay between catalytic domains linked to CBMs in LPMOs, and CAZymes in general.

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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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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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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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