|Formulation:||In 3.2 M ammonium sulphate|
|Stability:||> 4 years at 4oC|
|Synonyms:||cellulase; 4-beta-D-glucan 4-glucanohydrolase|
|Concentration:||Supplied at ~ 700 U/mL|
|Expression:||Purified from Trichoderma longibrachiatum|
|Specificity:||endo-hydrolysis of (1,4)-β-D-glucosidic linkages in cellulose.|
|Specific Activity:||~ 70 U/mg (40oC, pH 4.5 on CM-cellulose 4M)|
|Unit Definition:||One Unit of cellulase activity is defined as the amount of enzyme required to release one µmole of glucose reducing-sugar equivalents per minute from CM-cellulose 4M (10 mg/mL) in sodium acetate buffer (100 mM), pH 4.5 at 40oC.|
|Application examples:||Applications established in diagnostics and research within the textiles, food and feed, carbohydrate and biofuels industries.|
High purity Cellulase (endo-1,4-β-D-glucanase) (Trichoderma longibrachiatum) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Show complete list of our available Carbohydrate Active enZYmes.
(Aspergillus niger) E-CELBA - Cellulase (endo-1,4-β-D-glucanase)
(Bacillus amyloliquefaciens) E-CELTE - Cellulase (endo-1,4-β-D-glucanase)
(Talaromyces emersonii) E-CELTH - Cellulase (endo-1,4-β-D-glucanase)
(Thermobifida halotolerans) E-CELTM - Cellulase (endo-1,4-β-D-glucanase)
McCleary, B. V., McKie, V. & Draga, A. (2012). “Methods in Enzymology”, Volume 510, (H. Gilbert, Ed.), Elsevier Inc., pp. 1-17.
Several procedures are available for the measurement of endo-1,4-β-glucanase (EG). Primary methods employ defined oligosaccharides or highly purified polysaccharides and measure the rate of hydrolysis of glycosidic bonds using a reducing-sugar method. However, these primary methods are not suitable for the measurement of EG in crude fermentation broths due to the presence of reducing sugars and other enzymes active on these substrates. In such cases, dyed soluble or insoluble substrates are preferred as they are specific, sensitive, easy to use, and are not affected by other components, such as reducing sugars, in the enzyme preparation.Hide Abstract
McCleary, B. V. & Monaghan, D. (2000). “Proceedings of the Second European Symposium on Enzymes in Grain Processing”, (M. Tenkanen, Ed.), VTT Information Service, pp. 31-38.
Over the past 8 years, we have been actively involved in the development of simple and reliable assay procedures, for the measurement of enzymes of interest to the cereals and related industries. In some instances, different procedures have been developed for the measurement of the same enzyme activity (e.g. α-amylase) in a range of different materials (e.g. malt, cereal grains and fungal preparations). The reasons for different procedures may depend on several factors, such as the need for sensitivity, ease of use, robustness of the substrate mixture, or the possibility for automation. In this presentation, we will present information on our most up-to-date procedures for the measurement of α-amylase, endo-protease, β-glucanase and β-xylanase, with special reference to the use of particular assay formats in particular applications.Hide Abstract
Measurement of polysaccharide-degrading enzymes in plants using chromogenic and colorimetric substrates.
McCleary, B. V. (1995). “New Diagnostics in Crop Sciences”, (J. R. Skerritt and R. Appels, Eds.), CAB International, pp. 277-301.
Enzymatic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well-defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material (Bamforth, 1982). In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall-degrading enzymes are used to destroy the three-dimensional structure and water-binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate-degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial processes.Hide Abstract
Measurement of polysaccharide degrading enzymes using chromogenic and colorimetric substrates.
McCleary, B. V. (1991). Chemistry in Australia, September, 398-401.
Enzymic degradation of carbohydrates is of major significance in the industrial processing of cereals and fruits. In the production of beer, barley is germinated under well defined conditions (malting) to induce maximum enzyme synthesis with minimum respiration of reserve carbohydrates. The grains are dried and then extracted with water under controlled conditions. The amylolytic enzymes synthesized during malting, as well as those present in the original barley, convert the starch reserves to fermentable sugars. Other enzymes act on the cell wall polysaccharides, mixed-linkage β-glucan and arabinoxylan, reducing the viscosity and thus aiding filtration, and reducing the possibility of subsequent precipitation of polymeric material. In baking, β-amylase and α-amylase give controlled degradation of starch to fermentable sugars so as to sustain yeast growth and gas production. Excess quantities of α-amylase in the flour result in excessive degradation of starch during baking which in turn gives a sticky crumb texture and subsequent problems with bread slicing. Juice yield from fruit pulp is significantly improved if cell-wall degrading enzymes are used to destroy the three-dimensional structure and water binding capacity of the pectic polysaccharide components of the cell walls. Problems of routine and reliable assay of carbohydrate degrading enzymes in the presence of high levels of sugar compounds are experienced with such industrial process.Hide Abstract
Activity and Action of Cell-Wall Transglycanases.
Franková, L. & Fry, S. C. (2020). “The Plant Cell Wall”, Humana, New York, NY, pp. 165-192.
Transglycanases (endotransglycosylases) are enzymes that “cut and paste” polysaccharide chains. Several transglycanase activities have been discovered which can cut (i.e., use as donor substrate) each of the major hemicelluloses [xyloglucan, mannans, xylans, and mixed-linkage β-glucan (MLG)], and, as a recent addition, cellulose. These enzymes may play interesting roles in adjusting the wall’s physical properties, influencing cell expansion, stem strengthening, and fruit softening. Activities discussed include the homotransglycanases XET (xyloglucan endotransglucosylase, i.e., xyloglucan-xyloglucan endotransglycosylase), trans-β-mannanase (mannan-mannan endotransglycosylase), and trans-β-xylanase (xylan-xylan endotransglucosylase), plus the heterotransglycanases MXE (MLG-xyloglucan endotransglucosylase) and CXE (cellulose-xyloglucan endotransglucosylase). Transglycanases acting on polysaccharide donor substrates can utilize small, labeled oligosaccharides as acceptor substrates, generating easily recognizable polymeric labeled products. We present methods for extracting transglycanases from plant tissues and assaying them in vitro, either quantitatively in solution assays or by high-throughput dot-blot screens. Both radioactively and fluorescently labeled substrates are mentioned. A general procedure (glass-fiber blotting) is illustrated by which proposed novel transglycanase activities can be tested for. In addition, we describe strategies for detecting transglycanase action in vivo. These methods enable the quantification of, separately, XET and MXE action in Equisetum stems. Related methods enable the tissue distribution of transglycanase action to be visualized cytologically.Hide Abstract
Assembly of tomato fruit cuticles: a cross‐talk between the cutin polyester and cell wall polysaccharides.
Philippe, G., Geneix, N., Petit, J., Guillon, F., Sandt, C., Rothan, C., Lahaye, M., Marion, D. & Bakan, B. (2020). New Phytologist, 226(3), 809-822.
The cuticle is an essential and ubiquitous biological polymer composite covering aerial plant organs, whose structural component is the cutin polyester entangled with cell wall polysaccharides. The nature of the cutin‐embedded polysaccharides (CEPs) and their association with cutin polyester are still unresolved. Using tomato fruit as a model, chemical and enzymatic pretreatments combined with biochemical and biophysical methods were developed to compare the fine structure of CEPs with that of the noncutinized polysaccharides (NCPs). In addition, we used tomato fruits from cutin‐deficient transgenic lines cus1 (cutin synthase 1) to study the impact of cutin polymerization on the fine structure of CEPs. Cutin‐embedded polysaccharides exhibit specific structural features including a high degree of esterification (i.e. methylation and acetylation), a low ramification of rhamnogalacturonan (RGI), and a high crystallinity of cellulose. In addition to decreasing cutin deposition and polymerization, cus1 silencing induced a specific modification of CEPs, especially on pectin content, while NCPs were not affected. This new evidence of the structural specificities of CEPs and of the cross‐talk between cutin polymerization and polysaccharides provides new hypotheses concerning the formation of these complex lipopolysaccharide edifices.Hide Abstract
Cytosolic Acetyl-CoA generated by ATP-citrate lyase is essential for acetylation of cell wall polysaccharides.
Zhong, R., Cui, D., Richardson, E. A., Phillips, D. R., Azadi, P., Lu, G. & Ye, Z. H. (2020). Plant and Cell Physiology, 61(1), 64-75.
Plant cell wall polysaccharides, including xylan, glucomannan, xyloglucan and pectin, are often acetylated. Although a number of acetyltransferases responsible for the acetylation of some of these polysaccharides have been biochemically characterized, little is known about the source of acetyl donors and how acetyl donors are translocated into the Golgi, where these polysaccharides are synthesized. In this report, we investigated roles of ATP-citrate lyase (ACL) that generates cytosolic acetyl-CoA in cell wall polysaccharide acetylation and effects of simultaneous mutations of four Reduced Wall Acetylation (RWA) genes on acetyl-CoA transport into the Golgi in Arabidopsis thaliana. Expression analyses of genes involved in the generation of acetyl-CoA in different subcellular compartments showed that the expression of several ACL genes responsible for cytosolic acetyl-CoA synthesis was elevated in interfascicular fiber cells and induced by secondary wall-associated transcriptional activators. Simultaneous downregulation of the expression of ACL genes was demonstrated to result in a substantial decrease in the degree of xylan acetylation and a severe alteration in secondary wall structure in xylem vessels. In addition, the degree of acetylation of other cell wall polysaccharides, including glucomannan, xyloglucan and pectin, was also reduced. Moreover, Golgi-enriched membrane vesicles isolated from the rwa1/2/3/4 quadruple mutant were found to exhibit a drastic reduction in acetyl-CoA transport activity compared with the wild type. These findings indicate that cytosolic acetyl-CoA generated by ACL is essential for cell wall polysaccharide acetylation and RWAs are required for its transport from the cytosol into the Golgi.Hide Abstract
Exploring the action of endoglucanases on bleached eucalyptus kraft pulp as potential catalyst for isolation of cellulose nanocrystals.
Siqueira, G. A., Dias, I. K. & Arantes, V. (2019). International Journal of Biological Macromolecules, 133, 1249-1259.
Cellulose nanocrystals (CNCs) is a high-value and emerging bionanomaterial with an increasing number of applications. The action of endoglucanases (EGs) from fungal and bacterial sources belonging to three glycosyl hydrolase (GH) families were investigated on bleached eucalyptus kraft pulp as potential catalysts to prepare CNC. Fungal GH7EG was the most efficient in hydrolysis and fiber fragmentation without altering crystallinity and crystallite size. Fiber fragmentation promoted by fungal GH45EG was similar to that observed for GH7EG, although it released a smaller amount of sugar. Bacterial GH5EG resulted in very low hydrolysis yield and practically did not fragment the fibers, resulting in a hydrolysis residue with characteristics very similar to the initial material. GH45EG was the only EG that affected the crystallinity and crystallite size and also the only enzyme capable of isolating nanoparticles. The isolated nanoparticles had very narrow width distribution range of 6-10 nm and length distribution range of 400-600 nm. Supplementation of β-glucosidase and conventional mechanical refining as a pretreatment did not improve the release of nanoparticles. Despite catalyzing the same biochemical reaction, different EGs displayed very distinct action during hydrolysis. The reported strong binding of GH45EG's CBM to the cellulose and the lack of increased accessibility of the enzyme to new substrate likely allowed continuous hydrolysis of the few fibers available, resulting in the isolation of cellulose nanoparticles.Hide Abstract
Two new cellulolytic fungal species isolated from a 19th-century art collection.
Coronado-Ruiz, C., Avendaño, R., Escudero-Leyva, E., Conejo-Barboza, G., Chaverri, P. & Chavarría, M. (2018). Scientific Reports, 8(1), 7492.
The archive of the Universidad de Costa Rica maintains a nineteenth-century French collection of drawings and lithographs in which the biodeterioration by fungi is rampant. Because of nutritional conditions in which these fungi grew, we suspected that they possessed an ability to degrade cellulose. In this work our goal was to isolate and identify the fungal species responsible for the biodegradation of a nineteenth-century art collection and determine their cellulolytic activity. Fungi were isolated using potato-dextrose-agar (PDA) and water-agar with carboxymethyl cellulose (CMC). The identification of the fungi was assessed through DNA sequencing (nrDNA ITS and α-actin regions) complemented with morphological analyses. Assays for cellulolytic activity were conducted with Gram’s iodine as dye. Nineteen isolates were obtained, of which seventeen were identified through DNA sequencing to species level, belonging mainly to genera Arthrinium, Aspergillus, Chaetomium, Cladosporium, Colletotrichum, Penicillium and Trichoderma. For two samples that could not be identified through their ITS and α-actin sequences, a morphological analysis was conducted; they were identified as new species, named Periconia epilithographicola sp. nov. and Coniochaeta cipronana sp. nov. Qualitative tests showed that the fungal collection presents important cellulolytic activity.Hide Abstract
An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.
Sabbadin, F., Hemsworth, G. R., Ciano, L., Henrissat, B., Dupree, P., Tryfona, T., Marques, R. D. S., Sweeney, S. T., Besser, K., Elias, L., Pesante, G., Li, Y., Dowle, A. A., Bates, R., Gomez, L. D., Simister, R., Davies, G. J., Walton, P. H., Bruce, N. C. & Pesante, G. (2018). Nature communications, 9(1), 756.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over twenty members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin and was recruited by early arthropods with possible roles in remodelling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes towards cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.Hide Abstract
Cellodextrin phosphorylase from Ruminiclostridium thermocellum: X-ray crystal structure and substrate specificity analysis.
O'Neill, E. C., Pergolizzi, G., Stevenson, C. E., Lawson, D. M., Nepogodiev, S. A. & Field, R. A. (2017). Carbohydrate Research, 451, 118-132.
The GH94 glycoside hydrolase cellodextrin phosphorylase (CDP, EC 220.127.116.11) produces cellodextrin oligomers from short β-1→4-glucans and α-D-glucose 1-phosphate. Compared to cellobiose phosphorylase (CBP), which produces cellobiose from glucose and α-D-glucose 1-phosphate, CDP is biochemically less well characterised. Herein, we investigate the donor and acceptor substrate specificity of recombinant CDP from Ruminiclostridium thermocellum and we isolate and characterise a glucosamine addition product to the cellobiose acceptor with the non-natural donor α-D-glucosamine 1-phosphate. In addition, we report the first X-ray crystal structure of CDP, along with comparison to the available structures from CBPs and other closely related enzymes, which contributes to understanding of the key structural features necessary to discriminate between monosaccharide (CBP) and oligosaccharide (CDP) acceptor substrates.Hide Abstract
Xyloglucan gelation induced by enzymatic degalactosylation; kinetics and the effect of the molar mass.
Sakakibara, C. N., Sierakowski, M. R., Chassenieux, C., Nicolai, T. & de Freitas, R. A. (2017). Carbohydrate Polymers, 174, 517-523.
Gelation kinetics of aqueous solutions of xyloglucan (XG) extracted from H. courbaril seeds were investigated, in-situ, during enzymatic removal of galactose units by oscillatory shear rheological measurements, at different XG and enzyme (β-galactosidase) concentrations. Increasing the enzyme concentration (Cenz) led to an increase of the gelation rate. Master curves of the evolution of the storage shear modulus at different Cenz could be formed by time-Cenz superposition showing that Cenz influenced the kinetics, but not the gelation process and the final gel stiffness. The behaviour of gels formed by XG with different molar mass (Mw), prepared by endoglucanase hydrolysis, was evaluated as a function of the temperature. It was found that cooling led to a decrease of the crosslink density causing a decrease of the gel stiffness. The decrease of the crosslink density was sufficient to depercolate the network formed by relatively small XG with Mw = 105 g mol−1, but gels formed by XG with Mw ≥ 8 × 105 g mol−1 persisted down to 10°C. It is shown that the melting temperature and the gel stiffness at high temperatures can be controlled independently by varying the molar mass and the concentration of XG chains.Hide Abstract
Guillon, F., Moïse, A., Quemener, B., Bouchet, B., Devaux, M. F., Alvarado, C. & Lahaye, M. (2017). Plant Science, 257, 48-62.
Tomato fruit texture depends on histology and cell wall architecture, both under genetic and developmental controls. If ripening related cell wall modifications have been well documented with regard to softening, little is known about cell wall construction during early fruit development. Identification of key events and their kinetics with regard to tissue architecture and cell wall development can provide new insights on early phases of texture elaboration. In this study, changes in pectin and hemicellulose chemical characteristics and location were investigated in the pericarp tissue of tomato (Solanum lycopersicon var Levovil) at four stages of development (7, 14 and 21 day after anthesis (DPA) and mature green stages). Analysis of cell wall composition and polysaccharide structure revealed that both are continuously modified during fruit development. At early stages, the relative high rhamnose content in cell walls indicates a high synthesis of rhamnogalacturonan I next to homogalacturonan. Fine tuning of rhamnogalacturonan I side chains appears to occur from the cell expansion phase until prior to the mature green stage. Cell wall polysaccharide remodelling also concerns xyloglucans and (galacto)glucomannans, the major hemicelluloses in tomato cell walls. In situ localization of cell wall polysaccharides in pericarp tissue revealed non-ramified RG-I rich pectin and XyG at cellular junctions and in the middle lamella of young fruit. Blocks of non-methyl esterified homogalacturonan are detected as soon as 14 DPA in the mesocarp and remained restricted to cell corner and middle lamella whatever the stages. These results point to new questions about the role of pectin RGI and XyG in cell adhesion and its maintenance during cell expansion.Hide Abstract
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.Hide Abstract
Prakash, R., Hallett, I. C., Wong, S. F., Johnston, S. L., O’Donoghue, E. M., McAtee, P. A., Seal, A. G., Atkinson, R. G. & Schröder, R. (2017). BMC Plant Biology, 17(1), 86.
Background: Unlike in abscission or dehiscence, fruit of kiwifruit Actinidia eriantha develop the ability for peel detachment when they are ripe and soft in the absence of a morphologically identifiable abscission zone. Two closely-related genotypes with contrasting detachment behaviour have been identified. The ‘good-peeling’ genotype has detachment with clean debonding of cells, and a peel tissue that does not tear. The ‘poor-peeling’ genotype has poor detachability, with cells that rupture upon debonding, and peel tissue that fragments easily. Results: Structural studies indicated that peel detachability in both genotypes occurred in the outer pericarp beneath the hypodermis. Immunolabelling showed differences in methylesterification of pectin, where the interface of labelling coincided with the location of detachment in the good-peeling genotype, whereas in the poor-peeling genotype, no such interface existed. This zone of difference in methylesterification was enhanced by differential cell wall changes between the peel and outer pericarp tissue. Although both genotypes expressed two polygalacturonase genes, no enzyme activity was detected in the good-peeling genotype, suggesting limited pectin breakdown, keeping cell walls strong without tearing or fragmentation of the peel and flesh upon detachment. Differences in location and amounts of wall-stiffening galactan in the peel of the good-peeling genotype possibly contributed to this phenotype. Hemicellulose-acting transglycosylases were more active in the good-peeling genotype, suggesting an influence on peel flexibility by remodelling their substrates during development of detachability. High xyloglucanase activity in the peel of the good-peeling genotype may contribute by having a strengthening effect on the cellulose-xyloglucan network. Conclusions: In fruit of A. eriantha, peel detachability is due to the establishment of a zone of discontinuity created by differential cell wall changes in peel and outer pericarp tissues that lead to changes in mechanical properties of the peel. During ripening, the peel becomes flexible and the cells continue to adhere strongly to each other, preventing breakage, whereas the underlying outer pericarp loses cell wall strength as softening proceeds. Together these results reveal a novel and interesting mechanism for enabling cell separation.Hide Abstract
Zhou, Q., Ji, P., Zhang, J., Li, X. & Han, C. (2017). Journal of Bioscience and Bioengineering, 124(3), 271-276.
A novel thermostable endoglucanase (CTendo45) encoding gene was cloned from Chaetomium thermophilum and heterologously expressed in Pichia pastoris. Sequence alignment indicated that the CTendo45 enzyme belonged to glycoside hydrolase family 45. The recombinant enzyme was purified by Ni2+ affinity chromatography, and its apparent molecular mass was estimated to be 32 kDa by SDS-PAGE. The purified enzyme displayed maximum activity at 70°C and pH 4. CTendo45 was stable at 60°C for 1 h, and residual activities of 78.9% and 65.6% were estimated after 1 h at 70°C and 80°C, respectively. Ca2+, Zn2+, Mg2+, Cu2+ and Mn2+ were found to have beneficial effects on the enzyme activity to different degrees. The specific activity of purified CTendo45 was 1.52 IU mg-1 and the Km value was 59.6 µg ml-1 with a sodium carboxymethyl cellulose substrate. Moreover, CTendo45 exhibited high hydrolysis activity towards pectin, and the hydrolysis products were mainly galacturonic acid oligosaccharides. CTendo45 is the first reported bifunctional enzyme in glycoside hydrolase family 45 from C.thermophilum that is able to hydrolyze both cellulose and pectin. The biochemical properties of this recombinant CTendo45 make it a potentially effective glycoside hydrolase for industrial applications.Hide Abstract
Carter, R., Woolfenden, H., Baillie, A., Amsbury, S., Carroll, S., Healicon, E., Sovatzoglou, S., Braybrook, S., Gray, J. E., Hobbs, J., Morris, R. J. & Morris, R. J. (2017). Current Biology, 27(19), 2974-2983.
It has long been accepted that differential radial thickening of guard cells plays an important role in the turgor-driven shape changes required for stomatal pore opening to occur. This textbook description derives from an original interpretation of structure rather than measurement of mechanical properties. Here we show, using atomic force microscopy, that although mature guard cells display a radial gradient of stiffness, this is not present in immature guard cells, yet young stomata show a normal opening response. Finite element modeling supports the experimental observation that radial stiffening plays a very limited role in stomatal opening. In addition, our analysis reveals an unexpected stiffening of the polar regions of the stomata complexes, both in Arabidopsis and other plants, suggesting a widespread occurrence. Combined experimental data (analysis of guard cell wall epitopes and treatment of tissue with cell wall digesting enzymes, coupled with bioassay of guard cell function) plus modeling lead us to propose that polar stiffening reflects a mechanical, pectin-based pinning down of the guard cell ends, which restricts increase of stomatal complex length during opening. This is predicted to lead to an improved response sensitivity of stomatal aperture movement with respect to change of turgor pressure. Our results provide new insight into the mechanics of stomatal function, both negating an established view of the importance of radial thickening and providing evidence for a significant role for polar stiffening. Improved stomatal performance via altered cell-wall-mediated mechanics is likely to be of evolutionary and agronomic significance.Hide Abstract
Mühlmann, M., Kunze, M., Ribeiro, J., Geinitz, B., Lehmann, C., Schwaneberg, U., Commandeur, U. & Büchs, J. (2017). Journal of Biological Engineering, 11(1), 1.
Background: Cellulases are key player in the hydrolyzation of cellulose. Unfortunately, this reaction is slow and a bottleneck in the process chain from biomass to intermediates and biofuels due to low activities of the enzymes. To overcome this draw back, a lot of effort is put into the area of protein engineering, to modify these enzymes by directed evolution or rational design. Huge clone libraries are constructed and have to be screened for improved variants. High-throughput screening is the method of choice to tackle this experimental effort, but up to now only a few process steps are adapted to automated platforms and little attention has been turned to the reproducibility of clone rankings. Results: In this study, an extended robotic platform is presented to conduct automated high-throughput screenings of clone libraries including preculture synchronization and biomass specific induction. Automated upstream, downstream and analytical process steps are described and evaluated using E. coli and K. lactis as model organisms. Conventional protocols for media preparation, cell lysis, Azo-CMC assay and PAHBAH assay are successfully adapted to automatable high-throughput protocols. Finally, a recombinant E. coli celA2 clone library was screened and a reliable clone ranking could be realized. Conclusion: The RoboLector device is a suitable platform to perform all process steps of an automated high-throughput clone library screening for improved cellulases. On-line biomass growth measurement controlling liquid handling actions enables fair comparison of clone variants.Hide Abstract
Spier, V. C., Sierakowski, M. R., Ibrahim, A. T., Baum, J. C. S., Silveira, J. L. M. & de Freitas, R. A. (2015). International Journal of Biological Macromolecules, 81, 461-466.
Hydrolysis of xyloglucan from Tamarindus indica and Hymenaea courbaril seeds with endoglucanase (EGII), which randomly breaks the (1 → 4)-linked β-glycosidic bonds of the polymer chain, was monitored in real time using time-dependent viscometry analysis (TDV). For both samples there was a decrease in the intrinsic viscosity ([η]), viscosity average molar mass (Mv), radius de gyration (Rg ) and persistence length (Lp) immediately after the addition of the enzyme. It was observed the formation of oligosaccharides and oligomers composed of ~2 units, up to 140 min. Galactose-containing side chains two positions away from the non-substituted glucose, modulated the action of EGII, and the complete hydrolysis of the XG oligomers occurred after 24 h. The results demonstrate for the first time the real-time degradation of xyloglucan as well the macromolecular and oligosaccharide composition during the EGII hydrolysis process.Hide Abstract
de Freitas, R. A., Spier, V. C., Sierakowski, M. R., Nicolai, T., Benyahia, L. & Chassenieux, C. (2015). Carbohydrate Polymers, 129, 216-223.
Viscoelastic properties of aqueous solutions of xyloglucan extracted from Hymenaea courbaril seeds (Jatobá gum) were investigated by rheology over a wide range of concentrations and temperatures. The polymer was characterized in dilute solutions by light scattering measurements and size exclusion chromatography. Xyloglucan formed, in semi-dilute solutions (C 0.3 wt%), a transient network with cross-links characterized by a broad distribution of lifetimes, independent of the temperature and concentration. Progressively, at higher temperatures (>60°C), a second much weaker quasi-permanent network was formed and attributed to the exchange of intra- to inter-chain bonds. The stiffness of the second network increased with decreasing temperature, but it could be easily broken by applying a relatively weak shear stress and is readily reversible on re-heating, and partially reversible on resting at 20°C.Hide Abstract