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endo-1,4-β-Xylanase M1 (Trichoderma viride)

endo-1-4-beta-Xylanase M1 Trichoderma viride E-XYTR1
Product code: E-XYTR1
€156.00

8,000 Units

Prices exclude VAT

Available for shipping

Content: 8,000 Units
Shipping Temperature: Ambient
Storage Temperature: 2-8oC
Formulation: In 3.2 M ammonium sulphate
Physical Form: Suspension
Stability: > 4 years at 4oC
Enzyme Activity: endo-1,4-β-Xylanase
EC Number: 3.2.1.8
CAZy Family: GH11
CAS Number: 9025-57-4
Synonyms: endo-1,4-beta-xylanase; 4-beta-D-xylan xylanohydrolase
Source: Trichoderma viride
Molecular Weight: 20,500,
14,000
Concentration: Supplied at ~ 1,700 U/mL
Expression: Purified from Trichoderma viride
Specificity: endo-hydrolysis of (1,4)-β-D-xylosidic linkages in xylans.
Specific Activity: ~ 230 U/mg (40oC, pH 4.5 on wheat arabinoxylan)
Unit Definition: One Unit of xylanase activity is defined as the amount of enzyme required to release one µmole of xylose reducing-sugar equivalents per minute from wheat arabinoxylan (10 mg/mL) in sodium acetate buffer (100 mM), pH 4.5 at 40oC.
Temperature Optima: 50oC
pH Optima: 4.5
Application examples: Applications in carbohydrate and biofuels research and in the food and feeds and paper pulping industries.

High purity endo-1,4-β-Xylanase M1 (Trichoderma viride) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Documents
Certificate of Analysis
Safety Data Sheet
Booklet
Publications
Megazyme publication
Novel substrates for the automated and manual assay of endo-1,4-β-xylanase.

Mangan, D., Cornaggia, C., Liadova, A., McCormack, N., Ivory, R., McKie, V. A., Ormerod, A. & McCleary, D. V. (2017). Carbohydrate Research, 445, 14-22.

endo-1,4-β-Xylanase (EC 3.2.1.8) is employed across a broad range of industries including animal feed, brewing, baking, biofuels, detergents and pulp (paper). Despite its importance, a rapid, reliable, reproducible, automatable assay for this enzyme that is based on the use of a chemically defined substrate has not been described to date. Reported herein is a new enzyme coupled assay procedure, termed the XylX6 assay, that employs a novel substrate, namely 4,6-O-(3-ketobutylidene)-4-nitrophenyl-β-45-O-glucosyl-xylopentaoside. The development of the substrate and associated assay is discussed here and the relationship between the activity values obtained with the XylX6 assay versus traditional reducing sugar assays and its specificity and reproducibility were thoroughly investigated.

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Megazyme publication
Hydrolysis of wheat flour arabinoxylan, acid-debranched wheat flour arabinoxylan and arabino-xylo-oligosaccharides by β-xylanase, α-L-arabinofuranosidase and β-xylosidase.

McCleary, B. V., McKie, V. A., Draga, A., Rooney, E., Mangan, D. & Larkin, J. (2015). Carbohydrate Research, 407, 79-96.

A range of α-L-arabinofuranosyl-(1-4)-β-D-xylo-oligosaccharides (AXOS) were produced by hydrolysis of wheat flour arabinoxylan (WAX) and acid debranched arabinoxylan (ADWAX), in the presence and absence of an AXH-d3 α-L-arabinofuranosidase, by several GH10 and GH11 β-xylanases. The structures of the oligosaccharides were characterised by GC-MS and NMR and by hydrolysis by a range of α-L-arabinofuranosidases and β-xylosidase. The AXOS were purified and used to characterise the action patterns of the specific α-L-arabinofuranosidases. These enzymes, in combination with either Cellvibrio mixtus or Neocallimastix patriciarum β -xylanase, were used to produce elevated levels of specific AXOS on hydrolysis of WAX, such as 32-α-L-Araf-(1-4)-β-D-xylobiose (A3X), 23-α-L-Araf-(1-4)-β-D-xylotriose (A2XX), 33-α-L-Araf-(1-4)-β-D-xylotriose (A3XX), 22-α-L-Araf-(1-4)-β-D-xylotriose (XA2X), 32-α-L-Araf (1-4)-β-D-xylotriose (XA3X), 23-α-L-Araf-(1-4)-β-D-xylotetraose (XA2XX), 33-α-L-Araf-(1-4)-β-D-xylotetraose (XA3XX), 23 ,33-di-α-L-Araf-(1-4)-β-D-xylotriose (A2+3XX), 23,33-di-α-L-Araf-(1-4)-β-D-xylotetraose (XA2+3XX), 24,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA2+3XXX) and 33,34-di-α-L-Araf-(1-4)-β-D-xylopentaose (XA3A3XX), many of which have not previously been produced in sufficient quantities to allow their use as substrates in further enzymic studies. For A2,3XX, yields of approximately 16% of the starting material (wheat arabinoxylan) have been achieved. Mixtures of the α-L-arabinofuranosidases, with specific action on AXOS, have been combined with β-xylosidase and β-xylanase to obtain an optimal mixture for hydrolysis of arabinoxylan to L-arabinose and D-xylose.

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Megazyme publication
A Comparison of Polysaccharide Substrates and Reducing Sugar Methods for the Measurement of endo-1,4-β-Xylanase.

McCleary, B. V. & McGeough, P. (2015). Appl. Biochem. Biotechnol., 177(5), 1152-1163.

The most commonly used method for the measurement of the level of endo-xylanase in commercial enzyme preparations is the 3,5-dinitrosalicylic acid (DNS) reducing sugar method with birchwood xylan as substrate. It is well known that with the DNS method, much higher enzyme activity values are obtained than with the Nelson-Somogyi (NS) reducing sugar method. In this paper, we have compared the DNS and NS reducing sugar assays using a range of xylan-type substrates and accurately compared the molar response factors for xylose and a range of xylo-oligosaccharides. Purified beechwood xylan or wheat arabinoxylan is shown to be a suitable replacement for birchwood xylan which is no longer commercially available, and it is clearly demonstrated that the DNS method grossly overestimates endo-xylanase activity. Unlike the DNS assay, the NS assay gave the equivalent colour response with equimolar amounts of xylose, xylobiose, xylotriose and xylotetraose demonstrating that it accurately measures the quantity of glycosidic bonds cleaved by the endo-xylanase. The authors strongly recommend cessation of the use of the DNS assay for measurement of endo-xylanase due to the fact that the values obtained are grossly overestimated due to secondary reactions in colour development.

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Megazyme publication
A simple procedure for the large-scale purification of β-D-xylanase from Trichoderma viride.

Gibson, T. S. & McCleary, B. V. (1987). Carbohydrate Polymers, 7(3), 225- 240.

A simple procedure is described for the purification of gram quantities of β-D-xylanase from a commercially available Trichoderma viride culture filtrate. Chromatography of the crude extract on CM-Sepharose CL-6B gave two partially separated peaks of β-D-xylanase activity, and for convenience these have been termed xylanases I and II. Each has cellulase activity. The cellulase and xylanase activities were not separated by further purification on Ultrogel AcA 54 or Phenyl Sepharose CL-4B. Each of these xylanases was purified essentially to homogeneity (by the criterion of isoelectric focusing) and was free of protease, amylase and glycosidase activities. Physical and kinetic properties of xylanases I and II were identical, indicating that the separation of CM-Sepharose CL-6B may simply have been an artefact of chromatography. However, this pattern was reproducible, being obtained on several occasions. Each enzyme separated into two protein bands on isoelectric focusing. The major band had a pI of 8•45 and a very minor component had a pI of 7•3. Optimal activity was at pH 4•5 and 50°C and the enzymes were stable over a pH range of 3•4–7•9 and at temperatures below 55°C. Apparent Kms were 3•33 mg ml-1 on rye flour arabinoxylan and 1•33 mg ml-1 on larch wood xylan. The enzymes partially hydrolysed larch wood xylan to oligosaccharides with two or three D-xylosyl residues. Rye flour arabinoxylan was hydrolysed to high molecular weight oligosaccharides which were not fractionated on Bio-Gel P-2.

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Publication
New insights into the enzymatic hydrolysis of lignocellulosic polymers by using fluorescent tagged carbohydrate-binding modules.

Khatri, V., Meddeb-Mouelhi, F. & Beauregard, M. (2018). Sustainable Energy & Fuels, In Press.

The development of a bio-based economy requires the utilization of lignocellulosic biomass in a cost-effective way. The economic viability of lignocellulosic biomass-based industries is hindered by our imperfect understanding of biomass structure and suboptimal industrial processes. To achieve such goals requires direct and rapid monitoring of lignocellulosic polymers as they are physically, chemically, and/or enzymatically treated. In this study, the recently reported fluorescent protein tagged carbohydrate binding modules method (FTCM) was used to specifically track mechanical, chemical and enzymatic-induced variations of hemicelluloses at the surface of different wood fibers. Our results showed that susceptibility to hydrolysis in kraft pulp was higher for xylan, while mannan was more vulnerable in mechanical pulps. Furthermore, FTCM rapidly and efficiently detected enzymatic inactivation and the apparent complementarity (additive and/or synergistic effect) between cellulase and other enzymes (xylanase and mannanase), significantly bolstering cellulose and hemicelluloses hydrolysis. Subsequent addition of xylanase and mannanase enzymes directly proved that xylan was acting as a physical shield which was covering mannan in bleached kraft pulp. This study suggests that mannan was closely associated with cellulose or was deeply embedded in the cell wall organization of such fibers. FTCM provided direct support for previous models on fiber structure that were based on time-consuming and complicated approaches (i.e.chromatography, spectroscopy and microscopy). FTCM allowed for the monitoring of layers of polymers as they were exposed after treatments, providing key information regarding hydrolysis optimization and the specific susceptibility of xylan and mannan to biomass treatments. We believe that by applying this simple and rapid method on site, biomass industries could substantially improve cost-effectiveness of production of biofuels and other lignocellulosic biomass-based products.

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Publication
High activity CAZyme cassette for improving biomass degradation in thermophiles.

Brunecky, R., Chung, D., Sarai, N. S., Hengge, N., Russell, J. F., Young, J., Mittal, A., Pason, P., Vander Wall, T., Michener, W., Shollenberger, T., Westpheling, J. Himmel, M. E. & Bomble, Y. J. (2018). Biotechnology for Biofuels, 11(1), 22.

Background: Thermophilic microorganisms and their enzymes offer several advantages for industrial application over their mesophilic counterparts. For example, a hyperthermophilic anaerobe, Caldicellulosiruptor bescii, was recently isolated from hot springs in Kamchatka, Siberia, and shown to have very high cellulolytic activity. Additionally, it is one of a few microorganisms being considered as viable candidates for consolidated bioprocessing applications. Moreover, C. bescii is capable of deconstructing plant biomass without enzymatic or chemical pretreatment. This ability is accomplished by the production and secretion of free, multi-modular and multi-functional enzymes, one of which, CbCel9A/Cel48A also known as CelA, is able to outperform enzymes found in commercial enzyme preparations. Furthermore, the complete C. bescii exoproteome is extremely thermostable and highly active at elevated temperatures, unlike commercial fungal cellulases. Therefore, understanding the functional diversity of enzymes in the C. bescii exoproteome and how inter-molecular synergy between them confers C. bescii with its high cellulolytic activity is an important endeavor to enable the production of more efficient biomass degrading enzyme formulations and in turn, better cellulolytic industrial microorganisms. Results: To advance the understanding of the C. bescii exoproteome we have expressed, purified, and tested four of the primary enzymes found in the exoproteome and we have found that the combination of three or four of the most highly expressed enzymes exhibit synergistic activity. We also demonstrated that discrete combinations of these enzymes mimic and even improve upon the activity of the whole C. bescii exoproteome, even though some of the enzymes lack significant activity on their own. Conclusions: We have demonstrated that it is possible to replicate the cellulolytic activity of the native C. bescii exoproteome utilizing a minimal gene set, and that these minimal gene sets are more active than the whole exoproteome. In the future, this may lead to more simplified and efficient cellulolytic enzyme preparations or yield improvements when these enzymes are expressed in microorganisms engineered for consolidated bioprocessing.

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Publication
Laccase pretreatment for agrofood wastes valorization.

Giacobbe, S., Pezzella, C., Lettera, V., Sannia, G. & Piscitelli, A. (2018). Bioresource Technology, 265, 59-65.

Apple pomace, potato peels, and coffee silverskin are attractive agrofood wastes for the production of biofuels and chemicals, due to their abundance and carbohydrate content. As lignocellulosic biomasses, their conversion is challenged by the presence of lignin that prevents hydrolysis of polysaccharides, hence demanding a pretreatment step. In this work, the effectiveness of Pleurotus ostreatus laccases (with and without mediator) to remove lignin, improving the subsequent saccharification, was assessed. Optimized conditions for sequential protocol were set up for all agrofood wastes reaching delignification and detoxification yields correlated with high saccharification. Especially noteworthy were results for apple pomace and coffee silverskin for which 83% of and 73% saccharification yields were observed, by using laccase and laccase mediator system, respectively. The herein developed sequential protocol, saving soluble sugars and reducing the amount of wastewater, can improve the overall process for obtaining chemicals or fuels from agrofood wastes.

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Publication
Distribution of cell wall hemicelluloses in the wheat grain endosperm: a 3D perspective.

Fanuel, M., Ropartz, D., Guillon, F., Saulnier, L. & Rogniaux, H. (2018). Planta, 1-9.

Uneven distribution of AX and BG in lateral and longitudinal dimensions of a wheat grain was observed by three-dimensional MS imaging, presumably related to specific physicochemical properties of cell walls. Arabinoxylans (AX) and β-glucans (BG) are the main hemicelluloses that comprise the primary walls of starchy endosperm. These components are not evenly distributed in the endosperm, and the impact of their distribution on cell wall properties is not yet fully understood. Combined with on-tissue enzymatic degradation of the cell walls, mass spectrometry imaging (MSI) was used to monitor the molecular structure of AX and BG in thirty consecutive cross-sections of a mature wheat grain. A 3D image was built from the planar images, showing the distribution of these polymers at the full-grain level, both in lateral and longitudinal dimensions. BGs were more abundant at the vicinity of the germ and in the central cells of the endosperm, while AX, and especially highly substituted AX, were more abundant close to the brush and in the cells surrounding the crease (i.e., the transfer cells). Compared with the previously reported protocol, significant improvements were made in the tissue preparation to better preserve the shape of the fragile sections. This allowed to us achieve a good-quality 3D reconstruction from the consecutive 2D images. By providing a continuous view of the molecular distribution of the cell wall components across and along the grain, the three-dimensional images obtained by MSI may help understand the structure–function relationships of cell walls. The method should be readily extendable to other parietal polymers by selecting the appropriate enzymes.

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Publication
Pure enzyme cocktails tailored for the saccharification of sugarcane bagasse pretreated by using different methods.

Kim, I. J., Lee, H. J. & Kim, K. H. (2017). Process Biochemistry, 57, 167-174.

The compositions and physical properties of pretreated lignocellulose vary depending on pretreatment methods; therefore, enzyme cocktails specific to pretreatments are desired for efficient saccharification of lignocellulose. Here, enzyme cocktails consisting of three pure lignocellulolytic enzymes endoglucanase (EG), cellobiohydrolase (CBH) and endoxylanase (XN) with a fixed amount of β-glucosidase were tailored for acid- and alkali-pretreated sugarcane bagasse (ACID and ALKALI, respectively). Based on a mixture design, the optimal mass ratios of EG, CBH, and XN were determined to be 61.25:38.73:0.02 and 53.99:34.60:11.41 for ACID and ALKALI, respectively. The optimized enzyme cocktail yielded a higher or comparable amount of reducing sugars from the hydrolysis of ACID and ALKALI when compared to that obtained using commercial cellulase mixtures. Using the commercial and easily available pure enzymes, this simple method for the in-house preparation of an enzyme cocktail specific to pretreated lignocellulose consisting of only four enzymes with a high level of hydrolysis will be helpful for achieving enzymatic saccharification in the lignocellulose-based biorefinery.

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Publication
Multi-layer mucilage of Plantago ovata seeds: Rheological differences arise from variations in arabinoxylan side chains.

Yu, L., Yakubov, G. E., Zeng, W., Xing, X, Stenson, J., Bulone, V. &Stokes, J. R. (2017). Journal of the Science of Food and Agriculture, 165(1), 132-141.

Mucilages are hydrocolloid solutions produced by plants for a variety of functions, including the creation of a water-holding barrier around seeds. Here we report our discovery of the formation of three distinct mucilage layers around Plantago ovata seeds upon their hydration. Each layer is dominated by different arabinoxylans (AXs). These AXs are unusual because they are highly branched and contain β-1,3-linked xylose in their side chains. We show that these AXs have similar monosaccharide and linkage composition, but vary in their polymer conformation. They also exhibit distinct rheological properties in aqueous solution, despite analytical techniques including NMR showing little difference between them. Using enzymatic hydrolysis and chaotropic solvents, we reveal that hydrogen bonding and side chain distribution are key factors underpinning the distinct rheological properties of these complex AXs.

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Publication
Impact of different lignin fractions on saccharification efficiency in diverse species of the bioenergy crop miscanthus.

van der Weijde, T., Torres, A. F., Dolstra, O., Dechesne, A., Visser, R. G., & Trindade, L. M. (2016). BioEnergy Research, 9(1), 146-156.

Lignin is a key factor limiting saccharification of lignocellulosic feedstocks. In this comparative study, various lignin methods-including acetyl bromide lignin (ABL), acid detergent lignin (ADL), Klason lignin (KL), and modified ADL and KL determination methods—were evaluated for their potential to assess saccharification efficiency. Six diverse accessions of the bioenergy crop miscanthus were used for this analysis, which included accessions of Miscanthus sinensis, Miscanthus sacchariflorus, and hybrid species. Accessions showed large variation in lignin content. Lignin estimates were different between methods, but (highly) correlated to each other (0.54 ≤ r ≤ 0.94). The strength of negative correlations to saccharification efficiency following either alkaline or dilute acid pretreatment differed between lignin estimates. The strongest and most consistent correlations (−0.48 ≤ r ≤ −0.85) were obtained with a modified Klason lignin method. This method is suitable for high throughput analysis and was the most effective in detecting differences in lignin content (p < 0.001) between accessions.

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Publication
New insights into the structural and spatial variability of cell-wall polysaccharides during wheat grain development, as revealed through MALDI mass spectrometry imaging.

Veličković, D., Ropartz, D., Guillon, F., Saulnier, L. & Rogniaux, H. (2014). Journal of Experimental Botany, 65(8), 2079-2091.

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans (BG) are the major components of wheat grain cell walls. Although incompletely described at the molecular level, it is known that the chemical and distributional heterogeneity of these compounds impacts the quality and use of wheat. In this work, an emerging technique based on MALDI mass spectrometry imaging (MSI) was employed to map variations in the quantity, localization, and structure of these polysaccharides in the endosperm during wheat maturation. MALDI MSI couples detailed structural information with the spatial localization observed at the micrometer scale. The enzymic hydrolysis of AX and BG was performed directly on the grain sections, resulting in the efficient formation of smaller oligosaccharides that are easily measurable through MS, with no relocation across the grain. The relative quantification of the generated oligosaccharides was achieved. The method was validated by confirming data previously obtained using other analytical techniques. Furthermore, in situ analysis of grain cell walls through MSI revealed previously undetectable intense acetylation of AX in young compared to mature grains, together with findings concerning the feruloylation of AX and different structural features of BG. These results provide new insights into the physiological roles of these polysaccharides in cell walls and the specificity of the hydrolytic enzymes involved.

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Publication
Arabidopsis and Brachypodium distachyon transgenic plants expressing Aspergillus nidulans acetylesterases have decreased degree of polysaccharide acetylation and increased resistance to pathogens.

Pogorelko, G., Lionetti, V., Fursova, O., Sundaram, R. M., Qi, M., Whitham, S. A., Bogdanove, A. J., Bellincampi, D. & Zabotina, O. A. (2013). Plant Physiology, 162(1), 9-23.

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens.

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Publication
Evaluation of the xylan breakdown potential of eight mesophilic endoxylanases.

Cuyvers, S., Dornez, E., Moers, K., Pollet, A., Delcour, J. A. & Courtin, C. M. (2011). Enzyme and Microbial Technology, 49(3), 305-311.

In biomass degradation using simultaneous saccharification and fermentation (SSF), there is a need for efficient biomass degrading enzymes that can work at lower temperatures suitable for yeast fermentation. As xylan is an important lignocellulosic biomass constituent, this study aimed at investigating the possible differences in xylan breakdown potential of endoxylanases using eight different endoxylanases at conditions relevant for SSF. Both solubilising and degrading capacities of the endoxylanases were investigated using water-insoluble and water-soluble oat spelt xylan as model substrates for biomass xylan. Results showed that selecting for combinations of endoxylanases that are efficient at solubilising xylan on the one hand and degrading it to large extent on the other hand, coupled to high specific activities, seems the best option for complete xylan breakdown in lignocellulosic biomass conversion using SSF.

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Publication
Down-regulation of the CSLF6 gene results in decreased (1,3;1,4)-β-D-glucan in endosperm of wheat.

Nemeth, C., Freeman, J., Jones, H. D., Sparks, C., Pellny, T. K., Wilkinson, M. D., Dunwell, J., Andersson, A. A. M., Aman, P., Guillon, F., Saulnier, L., Mitchell, R. A. C. & Shewry, P. R. (2010). Plant Physiology, 152(3), 1209-1218.

(1,3;1,4)-β-D-Glucan (β-glucan) accounts for 20% of the total cell walls in the starchy endosperm of wheat (Triticum aestivum) and is an important source of dietary fiber for human nutrition with potential health benefits. Bioinformatic and array analyses of gene expression profiles in developing caryopses identified the CELLULOSE SYNTHASE-LIKE F6 (CSLF6) gene as encoding a putative β-glucan synthase. RNA interference constructs were therefore designed to down-regulate CSLF6 gene expression and expressed in transgenic wheat under the control of a starchy endosperm-specific HMW subunit gene promoter. Analysis of wholemeal flours using an enzyme-based kit and by high-performance anion-exchange chromatography after digestion with lichenase showed decreases in total β-glucan of between 30% and 52% and between 36% and 53%, respectively, in five transgenic lines compared to three control lines. The content of water-extractable β-glucan was also reduced by about 50% in the transgenic lines, and the Mr distribution of the fraction was decreased from an average of 79 to 85 X 104 g/mol in the controls and 36 to 57 X 104 g/mol in the transgenics. Immunolocalization of β-glucan in semithin sections of mature and developing grains confirmed that the impact of the transgene was confined to the starchy endosperm with little or no effect on the aleurone or outer layers of the grain. The results confirm that the CSLF6 gene of wheat encodes a β-glucan synthase and indicate that transgenic manipulation can be used to enhance the health benefits of wheat products.

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Publication
His374 of wheat endoxylanase inhibitor TAXI‐I stabilizes complex formation with glycoside hydrolase family 11 endoxylanases.

Fierens, K., Gils, A., Sansen, S., Brijs, K., Courtin, C. M., Declerck, P. J., De Ranter, C. J., Gebruers, K., Rabijns, A., Robben, J., Van Campenhout, S., Volckaert, G. & Delcour, J. A. (2005). FEBS Journal, 272(22), 5872-5882.

Wheat endoxylanase inhibitor TAXI-I inhibits microbial glycoside hydrolase family 11 endoxylanases. Crystallographic data of an Aspergillus niger endoxylanase-TAXI-I complex showed His374 of TAXI-I to be a key residue in endoxylanase inhibition [Sansen S, De Ranter CJ, Gebruers K, Brijs K, Courtin CM, Delcour JA & Rabijns A (2004) J Biol Chem 279, 36022–36028]. Its role in enzyme–inhibitor interaction was further investigated by site-directed mutagenesis of His374 into alanine, glutamine or lysine. Binding kinetics and affinities of the molecular interactions between A. niger, Bacillus subtilis, Trichoderma longibrachiatum endoxylanases and wild-type TAXI-I and TAXI-I His374 mutants were determined by surface plasmon resonance analysis. Enzyme–inhibitor binding was in accordance with a simple 1 : 1 binding model. Association and dissociation rate constants of wild-type TAXI-I towards the endoxylanases were in the range between 1.96 and 36.1 × 104m-1·s-1 and 0.72–3.60 × 10-4·s-1, respectively, resulting in equilibrium dissociation constants in the low nanomolar range. Mutation of TAXI-I His374 to a variable degree reduced the inhibition capacity of the inhibitor mainly due to higher complex dissociation rate constants (three- to 80-fold increase). The association rate constants were affected to a smaller extent (up to eightfold decrease). Substitution of TAXI-I His374 therefore strongly affects the affinity of the inhibitor for the enzymes. In addition, the results show that His374 plays a critical role in the stabilization of the endoxylanase–TAXI-I complex rather than in the docking of inhibitor onto enzyme.

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Nectarin IV, a potent endoglucanase inhibitor secreted into the nectar of ornamental tobacco plants. Isolation, cloning, and characterization.

Naqvi, S. M. S., Harper, A., Carter, C., Ren, G., Guirgis, A., York, W. S. & Thornburg, R. W. (2005). Plant Physiology, 139(3), 1389-1400.

We have isolated and characterized the Nectarin IV (NEC4) protein that accumulates in the nectar of ornamental tobacco plants (Nicotiana langsdorffii × Nicotiana sanderae var LxS8). This 60-kD protein has a blocked N terminus. Three tryptic peptides of the protein were isolated and sequenced using tandem mass spectroscopy. These unique peptides were found to be similar to the xyloglucan-specific fungal endoglucanase inhibitor protein (XEGIP) precursor in tomato (Lycopersicon esculentum) and its homolog in potato (Solanum tuberosum). A pair of oligonucleotide primers was designed based on the potato and tomato sequences that were used to clone a 1,018-bp internal piece of nec4 cDNA from a stage 6 nectary cDNA library. The remaining portions of the cDNA were subsequently captured by 5′ and 3′ rapid amplification of cDNA ends. Complete sequencing of the nec4 cDNA demonstrated that it belonged to a large family of homologous proteins from a wide variety of angiosperms. Related proteins include foliage proteins and seed storage proteins. Based upon conserved identity with the wheat (Triticum aestivum) xylanase inhibitor TAXI-1, we were able to develop a protein model that showed that NEC4 contains additional amino acid loops that are not found in TAXI-1 and that glycosylation sites are surface exposed. Both these loops and sites of glycosylation are on the opposite face of the NEC4 molecule from the site that interacts with fungal hemicellulases, as indicated by homology to TAXI-I. NEC4 also contains a region homologous to the TAXI-1 knottin domain; however, a deletion in this domain restructures the disulfide bridges of this domain, resulting in a pseudoknottin domain. Inhibition assays were performed to determine whether purified NEC4 was able to inhibit fungal endoglucanases and xylanases. These studies showed that NEC4 was a very effective inhibitor of a family GH12 xyloglucan-specific endoglucanase with a Ki of 0.35 nM. However, no inhibitory activity was observed against other family GH10 or GH11 xylanases. The patterns of expression of the NEC4 protein indicate that, while expressed in nectar at anthesis, it is most strongly expressed in the nectary gland after fertilization, indicating that inhibition of fungal cell wall-degrading enzymes may be more important after fertilization than before.

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Endoxylanase substrate selectivity determines degradation of wheat water-extractable and water-unextractable arabinoxylan.

Moers, K., Celus, I., Brijs, K., Courtin, C. M. & Delcour, J. A. (2005). Carbohydrate Research, 340(7), 1319-1327.

The relative activity of an endoxylanase towards water-unextractable (WU-AX) and water-extractable arabinoxylan (WE-AX) substrates, referred to as endoxylanase substrate selectivity, impacts the enzyme functionality in cereal-based biotechnological processes such as bread-making and gluten starch separation. A set of six endoxylanases representing a range of substrate selectivities as determined by a screening method using chromophoric substrates [Anal. Biochem. 2003, 319, 73–77] was used to examine the impact of such selectivity on changes in structural characteristics of wheat WU-AX and WE-AX upon enzymic hydrolysis. While WE-AX degradation by the selected endoxylanases was very comparable with respect to apparent molecular mass (MM) profiles and arabinose to xylose ratio of the hydrolysates formed, WU-AX solubilisation and subsequent degradation of solubilised fragments gave rise to widely varying MM profiles, depending on the substrate selectivity of the enzymes. Enzymes with high selectivity towards WU-AX de facto generated higher MM fragments from WU-AX than enzymes with low selectivity. The arabinose to xylose ratios of solubilised fragments were independent of the degree of solubilisation.

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Hazard Statements : Not Applicable
Precautionary Statements : Not Applicable
Safety Data Sheet
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