|Stability:||> 10 years under recommended storage conditions|
|Main Chain Glycosidic Linkage:||β-1,4|
|Substrate For (Enzyme):||endo-Cellulase|
This product has been discontinued (read more).
High purity Carboxymethyl Cellulose 4M for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Carboxymethylated (DS ~ 0.4), medium viscosity cellulose. Recommended substrate for the assay of cellulases by reducing sugar methods. For most endo-cellulases, this is a much more sensitive substrate than CMC-7M (DS = 0.7).
View our extensive polysaccharides product list.
Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species.
Li, J., Solhi, L., Goddard-Borger, E. D., Mathieu, Y., Wakarchuk, W. W., Withers, S. G. & Brumer, H. (2021). Biotechnology for Biofuels, 14(1), 1-19.
Background: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.Hide Abstract
Novel multi‐domain, multi‐functional glycoside hydrolases from highly lignocellulolytic Caldicellulosiruptor species.
Conway, J. M., Crosby, J. R., Hren, A. P., Southerland, R. T., Lee, L. L., Lunin, V. V., Alahuhta, P., Himmel, M. E., Bomble, Y. J., Adams, M. W. W. & Kelly, R. M. (2018). AIChE Journal, 64(12), 4218-4228.
Biological hydrolysis of microcrystalline cellulose is an uncommon feature in the microbial world, especially among bacteria and archaea growing optimally above 70°C (the so‐called extreme thermophiles). In fact, among this group only certain species in the genus Caldicellulosiruptor are capable of rapid and extensive cellulose degradation. Four novel multi‐domain glycoside hydrolases (GHs) from Caldicellulosiruptor morganii and Caldicellulosiruptor danielii were produced recombinantly in Caldicellulosiruptor bescii and characterized. These GHs are structurally organized with two or three catalytic domains flanking carbohydrate binding modules from Family 3 (CBM3). Collectively, these enzymes represent GH families 5, 9, 10, 12, 44, 48, and 74, and hydrolyze crystalline cellulose, glucan, xylan, and mannan, the primary carbohydrates in plant biomass. Degradation of microcrystalline cellulose by cocktails of GHs from three Caldicellulosiruptor species demonstrated that synergistic interactions enable mixtures of multiple enzymes to outperform single enzymes, suggesting a community mode of action for lignocellulose utilization in thermal environments.Hide Abstract
Salmeán, A. A., Guillouzo, A., Duffieux, D., Jam, M., Matard-Mann, M., Larocque, R., Pedersen, H. L., Michel, G., Czjzek, M., Willats, W. G. T. & Hervé, C. (2018). Scientific Reports, 8(1), 2500.
Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources.Hide Abstract
Pires, V. M. R., Pereira, P. M. M., Brás, J. L. A., Correia, M., Cardoso, V., Bule, P., Alves, V. D., Najmudin, S., Venditto, I., Ferreira, L. M. A., Romão, M. J., Carvalho, A. L., Fontes, C. M. G. A. & Romão, M. J. (2017). Journal of Biological Chemistry, 292(12), 4847-4860.
Deconstruction of cellulose, the most abundant plant cell wall polysaccharide, requires the cooperative activity of a large repertoire of microbial enzymes. Modular cellulases contain non-catalytic type A carbohydrate-binding modules (CBMs) that specifically bind to the crystalline regions of cellulose, thus promoting enzyme efficacy through proximity and targeting effects. Although type A CBMs play a critical role in cellulose recycling, their mechanism of action remains poorly understood. Here we produced a library of recombinant CBMs representative of the known diversity of type A modules. The binding properties of 40 CBMs, in fusion with an N-terminal GFP domain, revealed that type A CBMs possess the ability to recognize different crystalline forms of cellulose and chitin over a wide range of temperatures, pH levels, and ionic strengths. A Spirochaeta thermophila CBM64, in particular, displayed plasticity in its capacity to bind both crystalline and soluble carbohydrates under a wide range of extreme conditions. The structure of S. thermophila StCBM64C revealed an untwisted, flat, carbohydrate-binding interface comprising the side chains of four tryptophan residues in a co-planar linear arrangement. Significantly, two highly conserved asparagine side chains, each one located between two tryptophan residues, are critical to insoluble and soluble glucan recognition but not to bind xyloglucan. Thus, CBM64 compact structure and its extended and versatile ligand interacting platform illustrate how type A CBMs target their appended plant cell wall-degrading enzymes to a diversity of recalcitrant carbohydrates under a wide range of environmental conditions.Hide Abstract
A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomics.
Patrascu, O., Béguet-Crespel, F., Marinelli, L., Le Chatelier, E., Abraham, A., Leclerc, M., Klopp, C., Terrapon, N., Henrissat, B., Blottière, H. M., Doré, J. & Christel Béra-Maillet. (2017). Scientific Reports, 7, 40248.
The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.Hide Abstract
Cheng, L., Duan, S., Feng, X., Zheng, K., Yang, Q. & Liu, Z. (2016). BioMed Research International, 2016, In Press.
β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+, and strongly inhibited by Ba2+, and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 µmol/min/mL versus 1.749 mg/mL and 33.45 µ mol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.Hide Abstract
Takata, R., Tokita, K., Mori, S., Shimoda, R., Harada, N., Ichinose, H., Kaneko, A., Igarashi, k., Samejima, M., Tsumuraya, Y. & Kotake, T. (2010). Carbohydrate Research, 345(17), 2516-2522.
Arabinogalactan-proteins (AGPs) are a family of plant proteoglycans having large carbohydrate moieties attached to core-proteins. The carbohydrate moieties of AGPs commonly have β-(1→3)(1→6)-galactan as the backbone, to which other auxiliary sugars such as L-Ara and GlcA are attached. For the present study, an α-L-arabinofuranosidase belonging to glycoside hydrolase family (GHF) 54, NcAraf1, and an endo-β-(1→6)-galactanase of GHF 5, Nc6GAL, were identified in Neurospora crassa. Recombinant NcAraf1 (rNcAraf1) expressed in Pichia pastoris hydrolyzed radish AGPs as well as arabinan and arabinoxylan, showing relatively broad substrate specificity toward polysaccharides containing α-L-arabinofuranosyl residues. Recombinant Nc6GAL (rNc6GAL) expressed in P. pastoris specifically acted on β-(1→6)-galactosyl residues. Whereas AGP from radish roots was hardly hydrolyzed by rNc6GAL alone, β-(1→6)-galactan side chains were reduced to one or two galactan residues by a combination of rNcAraf1 and rNc6GAL. These results suggest that the carbohydrate moieties of AGPs are degraded by the concerted action of NcAraf1 and Nc6GAL secreted from N. crassa.Hide Abstract
Wong, K. H. & Cheung, P. C. K. (2009). Food Chemistry, 115(3), 795-800.
A comparative study on preparing dietary fibres (DFs) from three mushroom sclerotia, namely, Pleurotus tuber-regium (PTR), Polyporus rhinocerus (PR) and Wolfiporia cocos (WC), using analytical or industrial enzymes (including α-amylase, protease and amyloglucosidase), was conducted. Apart from enzyme activity and purity, their effects on the yield of sclerotial DF as well as its major components, such as β-glucans, chitin and resistant glycogen (RG), were investigated and compared. The activities of all industrial enzymes were significantly lower than those of their corresponding analytical ones, except for the Fungamyl® Super MA, which had the highest α-amylase activity (6395 U/g). However, this fungal α-amylase was less able to digest the three sclerotial glycogens when compared with the bacterial alternatives. Amongst all tested enzymes, only analytical and industrial amyloglucosidases were found to have significant amount of contaminating cellulase (7.05–7.07 U/ml) and lichenase (4.62–4.67 U/ml) activities, which would cause endo-depolymerization of the β-glucan-type cell wall components (3.39% reduction in glucose residue after RG correction) of the PTR, leading to a marked α-amylase hydrolysis of its otherwise physically-inaccessible cytoplasmic glycogen (20.3% reduction in RG content). Commercial production of the three novel sclerotial DFs, using the industrial enzymes, would be feasible since, in addition to their economic advantage, both the yield (PTR: 81.2%; PR: 86.5%; WC: 96.2% of sample DM) and total non-starch polysaccharide contents (PTR: 88.0%; PR: 92.5%; WC: 91.1% DF-rich materials of DM) of their resulting sclerotial DFs were comparable to the levels of those prepared using analytical enzymes.Hide Abstract
Konishi, T., Kotake, T., Soraya, D., Matsuoka, K., Koyama, T., Kaneko, S., Igarashi, K., Samejima, M. & Tsumuraya, Y. (2008). Carbohydrate Research, 343(7), 1191-1201.
The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, L-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 β-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native β-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP β-GlcA occurred at pH 3.0–4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent Km values of rAnGlcAase were 30.4 µM for PNP β-GlcA and 422µM for β-GlcA-(1→6)-Gal, and those of rNcGlcAase were 38.3 µM and 378 µM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP β-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of β-glucuronidase with the capacity to hydrolyze β-GlcA and 4-Me-β-GlcA residues of AGPs, although they differ significantly in their preferences.Hide Abstract
A xyloglucan-specific family 12 glycosyl hydrolase from Aspergillus niger: recombinant expression, purification and characterization.
Master, E. R., Zheng, Y., Storms, R., Tsang, A. & Powlowski, J. (2008). Biochem. J, 411, 161-170.
A new GH12 (glycosyl hydrolase 12) family XEG [xyloglucan-specific endo-β-1,4-glucanase (EC 184.108.40.206)] from Aspergillus niger, AnXEG12A, was overexpressed, purified and characterized. Whereas seven xyloglucanases from GH74 and two xyloglucanases from GH5 have been characterized previously, this is only the third characterized example of a GH12 family xyloglucanase. GH12 enzymes are structurally and mechanistically distinct from GH74 enzymes. Although over 100 GH12 sequences are now available, little is known about the structural and biochemical bases of xyloglucan binding and hydrolysis by GH12 enzymes. Comparison of the AnXEG12A cDNA sequence with the genome sequence of A. niger showed the presence of two introns, one in the coding region and the second one in the 333-nt-long 3´-untranslated region of the transcript. The enzyme was expressed recombinantly in A. niger and was readily purified from the culture supernatant. The isolated enzyme appeared to have been processed by a kexin-type protease, which removed a short prosequence. The substrate specificity was restricted to xyloglucan, with cleavage at unbranched glucose in the backbone. The apparent kinetic parameters were similar to those reported for other xyloglucan-degrading endoglucanases. The pH optimum (5.0) and temperature resulting in highest enzyme activity (50–60°C) were higher than those reported for a GH12 family xyloglucanase from Aspergillus aculeatus, but similar to those of cellulose-specific endoglucanases from the GH12 family. Phylogenetic, sequence and structural comparisons of GH12 family endoglucanases helped to delineate features that appear to be correlated to xyloglucan specificity.Hide Abstract
Martinez-Fleites, C., Guerreiro, C. I. P.D., Baumann, M. J., Taylor, E. J., Prates, J. A. M., Ferreira, L. M. A., Fontes, C. M. G. A., Brumer, H. & Davies, G. J. (2006). Journal of Biological Chemistry, 281(34), 24922-24933.
The enzymatic degradation of the plant cell wall is central both to the natural carbon cycle and, increasingly, to environmentally friendly routes to biomass conversion, including the production of biofuels. The plant cell wall is a complex composite of cellulose microfibrils embedded in diverse polysaccharides collectively termed hemicelluloses. Xyloglucan is one such polysaccharide whose hydrolysis is catalyzed by diverse xyloglucanases. Here we present the structure of the Clostridium thermocellum xyloglucanase Xgh74A in both apo and ligand-complexed forms. The structures, in combination with mutagenesis data on the catalytic residues and the kinetics and specificity of xyloglucan hydrolysis reveal a complex subsite specificity accommodating seventeen monosaccharide moieties of the multibranched substrate in an open substrate binding terrain.Hide Abstract
Molecular cloning and expression in Escherichia coli of a Trichoderma viride endo-beta-(1→6)-galactanase gene.
Kotake, T., Kaneko, S., Kubomoto, A., Haque, M., Kobayashi, H. & Tsumuraya, Y. (2004). Biochem. J, 377(3), 749-755.
A gene encoding endo-β-(1→6)-galactanase from Trichoderma viride was cloned by reverse transcriptase–PCR and expressed in Escherichia coli. The gene contained an open reading frame consisting of 1437 bp (479 amino acids). The deduced amino acid sequence of the protein showed little similarity with other known glycoside hydrolases. A signal sequence (20 amino acids) was found at the N-terminal region of the protein and the molecular mass of the mature form was calculated to be 50.488 kDa. The gene product expressed in E. coli as a recombinant protein fused with thioredoxin and His6 tags had almost the same substrate specificity and mode of action as native enzyme purified from a commercial cellulase preparation of T. viride, i.e. recombinant enzyme endo-hydrolysed β-(1→6)-galacto-oligomers with a DP (degree of polymerization) higher than 3, and it could also hydrolyse α-L-arabinofuranosidase-treated arabinogalactan protein from radish. It produced β-(1→6)-galacto-oligomers ranging from DP 2 to at least 8 at the initial hydrolysis stage and galactose and β-(1→6)-galactobiose as the major products at the final reaction stage. These results indicate that the cloned gene encodes an endo-β-(1→6)-galactanase. As far as we know, this is the first time an endo-β-(1→6)-galactanase has been cloned.Hide Abstract
Mølhøj, M., Ulvskov, P. & Dal Degan, F. (2001). Plant Physiology, 127(2), 674-684.
The Brassica napus gene, Cel16, encodes a membrane-anchored endo-1,4-β-glucanase with a deduced molecular mass of 69 kD. As for other membrane-anchored endo-1,4-β-glucanases, Cel16 consists of a predicted intracellular, charged N terminus (methionine1-lysine70), a hydrophobic transmembrane domain (isoleucine71-valine93), and a periplasmic catalytic core (lysine94-proline621). Here, we report the functional analysis of Δ1-90Cel16 , the N terminally truncated Cel16, missing residues 1 through 90 and comprising the catalytic domain of Cel16 expressed recombinantly in the methylotrophic yeast Pichia pastoris as a soluble protein. A two-step purification protocol yielded Δ1-90Cel16 in a pure form. The molecular mass of Δ1-90Cel16, when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was about 130 kD and about 60 kD after enzymatic removal of N-glycans, fitting the expected molecular mass of 59 kD. Δ1-90Cel16 was highly N glycosylated as compared with the native B. napus Cel16 protein. Δ1-90Cel16 had a pH optimum of 6.0. The activity of Δ1-90Cel16 was inhibited by EDTA and exhibited a strong dependence on calcium. Δ1-90Cel16 showed substrate specificity for low substituted carboxymethyl-cellulose and amorphous cellulose. It did not hydrolyze crystalline cellulose, xyloglycan, xylan, (1→3),(1→4)-β-D-glucan, the highly substituted hydroxyethylcellulose, or the oligosaccharides cellotriose, cellotetraose, cellopentaose, or xylopentaose. Size exclusion analysis of Δ1-90Cel16-hydrolyzed carboxymethylcellulose showed that Δ1-90Cel16 is a true endo-acting glucanase.Hide Abstract