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|Storage Temperature:||Below -10oC|
|Stability:||> 10 years under recommended storage conditions|
|Substrate For (Enzyme):||endo-Chitinase|
High purity Hexaacetyl-chitohexaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Prepared from chitin.
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.Hide Abstract
Krolicka, M., Hinz, S. W., Koetsier, M. J., Eggink, G., van den Broek, L. A. & Boeriu, C. G. (2018). Applied Microbiology and Biotechnology, 102(17), 7441-7454.
Thermostable enzymes are a promising alternative for chemical catalysts currently used for the production of N-acetylglucosamine (GlcNAc) from chitin. In this study, a novel thermostable β-N-acetylglucosaminidase MthNAG was cloned and purified from the thermophilic fungus Myceliophthora thermophila C1. MthNAG is a protein with a molecular weight of 71 kDa as determined with MALDI-TOF-MS. MthNAG has the highest activity at 50°C and pH 4.5. The enzyme shows high thermostability above the optimum temperature: at 55°C (144 h, 75% activity), 60°C (48 h, 85% activity; half-life 82 h), and 70°C (24 h, 33% activity; half-life 18 h). MthNAG releases GlcNAc from chitin oligosaccharides (GlcNAc)2-5, p-nitrophenol derivatives of chitin oligosaccharides (GlcNAc)1-3-pNP, and the polymeric substrates swollen chitin and soluble chitosan. The highest activity was detected towards (GlcNAc)2. MthNAG released GlcNAc from the non-reducing end of the substrate. We found that MtHNAG and Chitinase Chi1 from M. thermophila C1 synergistically degraded swollen chitin and released GlcNAc in concentration of approximately 130 times higher than when only MthNAG was used. Therefore, chitinase Chi1 and MthNAG have great potential in the industrial production of GlcNAc.Hide Abstract
Characterization and synergistic action of a tetra‐modular lytic polysaccharide monooxygenase from Bacillus cereus.
Mutahir, Z., Mekasha, S., Loose, J. S., Abbas, F., Vaaje‐Kolstad, G., Eijsink, V. G. & Forsberg, Z. (2018). FEBS Letters, 592, 2562-2571.
Lytic polysaccharide monooxygenases (LPMOs) contribute to enzymatic conversion of recalcitrant polysaccharides such as chitin and cellulose and may also play a role in bacterial infections. Some LPMOs are multimodular, the implications of which remain only partly understood. We have studied the properties of a tetra‐modular LPMO from the food poisoning bacterium Bacillus cereus (named BcLPMO10A). We show that BcLPMO10A, comprising an LPMO domain, two fibronectin‐type III (FnIII)‐like domains, and a carbohydrate‐binding module (CBM5), is a powerful chitin‐active LPMO. While the role of the FnIII domains remains unclear, we show that enzyme functionality strongly depends on the CBM5, which, by promoting substrate binding, protects the enzyme from inactivation. BcLPMO10A enhances the activity of chitinases during the degradation of α-chitin.Hide Abstract
Mitsuya, D., Sugiyama, T., Zhang, S., Takeuchi, Y., Okai, M., Urano, N. & Ishida, M. (2018). Journal of Bioscience and Bioengineering, 126(2), 169-175.
We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.Hide Abstract
Li, K., Chen, W., Wang, W., Tan, H., Li, S. & Yin, H. (2018). Carbohydrate Polymers, 201, 122-130.
Curdlan is a water-insoluble microbial exo-polysaccharide that is hardly degraded. The gene CcGluE encoding an endo-β-1 →3-glucanase consisting of 412 amino acids (44 kDa) from Cellulosimicrobium cellulans E4-5 was cloned and expressed in Escherichia coli. The recombinant CcGluE hydrolysed curdlan powder effectively. CcGluE shows high endo-β-1 →3 glucanase activity and low β-1,4 and β-1,6 glucanase activities with broad substrate specificity for glucan, including curdlan, laminarin and β-1 →3/1 →6-glucan, and the highest catalytic activity for curdlan. Moreover, the hydrolytic products of curdlan were glucan oligosaccharides with degrees of polymerisation of 2-13, and the main products were glucobiose and glucotriose. Degradation mode analysis indicated that CcGluE is more likely to hydrolyse glucopentaose and revealed that CcGluE was an endo-glucanase. Furthermore, upon combination with a homogenising pre-treatment method with curdlan, the degradation efficiency of CcGluE for curdlan powder was greatly improved 7.1-fold, which laid a good foundation for the utilisation of curdlan.Hide Abstract
Nareddy, P. K. & Swamy, M. J. (2017). International Journal of Biological Macromolecules, In Press.
In plants, chitooligosaccharide-binding phloem exudate lectins play an important role in the defense mechanism against parasites. Here, we investigated the thermal and chaotrope-induced unfolding of cucumber (Cucumis sativus) phloem exudate lectin (CPL). Circular dichroism (CD) spectroscopic studies indicate that the secondary and tertiary structures of CPL are essentially unaltered up to 90°C. Consistent with this, differential scanning calorimetric studies revealed that CPL is highly thermostable and undergoes a cooperative thermal unfolding transition centered at 97.6°C. The unfolding process was calorimetrically irreversible, and could be described by a non-two-state model, suggesting that upon undergoing a reversible unfolding transition the protein attains a final state in an irreversible step. The ratio of calorimetric and van’t Hoff enthalpies (ΔHc/ΔHv/) was >1.0, suggesting that the two monomers in the dimeric protein unfold at the same temperature. CD spectra recorded at different pH indicated that the secondary and tertiary structures of the protein are nearly unaltered in the pH range 3.0-10.0. Guanidine hydrochloride-induced unfolding studies indicate that chemical denaturation of CPL can also be described by a two-state process, without involving any intermediate. The stability of CPL to high temperatures and large variations of pH appear to be particularly suited for its role in plant defense.Hide Abstract
Moon, C., Seo, D. J., Song, Y. S., Hong, S. H., Choi, S. H. & Jung, W. J. (2017). Microbial Pathogenesis, 113, 218-224.
Serratia marcescens PRNK-1, which has strong chitinolytic activity, was isolated from cockroaches (Periplaneta Americana L.). The chitinase from S. marcescens PRNK-1 was characterized after incubation in a 0.5% colloidalchitin medium at 30°C for 3 days. The molecular weights of three bands after staining for chitinase activity were approximately 34, 41, and 48 kDa on an SDS-PAGE gel. S. marcescens PRNK-1 strain strongly inhibited hyphal growth of Rhizoctonia solani and Fusarium oxysporum. Thin-layer chromatography(TLC) and high performance liquid chromatograph (HPLC) analyses were conducted to investigate the degradation patterns of N-acetyl-chitooligosaccharides by PRNK-1 chitinase. The N-acetyl-chitooligosaccharides: N-acetyl-chitin dimer (GlcNAc)2, N-acetyl-chitin trimer (GlcNAc)3, and N-acetyl-chitin tetramer (GlcNAc)4 were degraded to (GlcNAc)1-3 on a TLC plate. In an additional experiment, (GlcNAc)6 was degraded to (GlcNAc)1-4 on a TLC plate. The optimal temperature for chitinase activity of the PRNK-1 was 50°C, producing 32.8 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred with 50°C incubation. The optimal pH for chitinase activity of PRNK-1 was pH 5.5, producing 24.6 units/mL. As seen via TLC, the highest degradation of (GlcNAc)4 by PRNK-1 chitinase occurred at pH 5.0-6.0. These results indicate that chitinase produced from S. marcescens PRNK-1 strain showed strong antifungal activity and potential of production of N-acetyl-chitooligosaccharides.Hide Abstract
Regel, E. K., Weikert, T., Niehues, A., Moerschbacher, B. M. & Singh, R. (2017). Biotechnology and Bioengineering, In Press.
Partially acetylated chitosan oligosaccharides (paCOS) have various potential applications in agriculture, biomedicine and pharmaceutics due to their suitable bioactivities. One method to produce paCOS is partial chemical hydrolysis of chitosan polymers, but that leads to poorly defined mixtures of oligosaccharides. However, the effective production of defined paCOS is crucial for fundamental research and for developing applications. A more promising approach is enzymatic depolymerization of chitosan using chitinases or chitosanases, as the substrate specificity of the enzyme determines the composition of the oligomeric products. Protein-engineering of these enzymes to alter their substrate specificity can overcome the limitations associated with naturally occurring enzymes and expand the spectrum of specific paCOS that can be produced. Here, engineering the substrate specificity of Bacillus sp. MN chitosanase is described for the first time. Two muteins with active site substitutions can accept N-acetyl-D-glucosamine units at their subsite (-2), which is impossible for the wildtype enzyme.Hide Abstract
Tuveng, T. R., Rothweiler, U., Udatha, G., Vaaje-Kolstad, G., Smalås, A. & Eijsink, V. G. (2017). PloS One, 12(11), e0187544.
Chitin, a polymer of β(1–4)-linked N-acetylglucosamine found in e.g. arthropods, is a valuable resource that may be used to produce chitosan and chitooligosaccharides, two compounds with considerable industrial and biomedical potential. Deacetylating enzymes may be used to tailor the properties of chitin and its derived products. Here, we describe a novel CE4 enzyme originating from a marine Arthrobacter species (ArCE4A). Crystal structures of this novel deacetylase were determined, with and without bound chitobiose [(GlcNAc)2], and refined to 2.1 Å and 1.6 Å, respectively. In-depth biochemical characterization showed that ArCE4A has broad substrate specificity, with higher activity against longer oligosaccharides. Mass spectrometry-based sequencing of reaction products generated from a fully acetylated pentamer showed that internal sugars are more prone to deacetylation than the ends. These enzyme properties are discussed in the light of the structure of the enzyme-ligand complex, which adds valuable information to our still rather limited knowledge on enzyme-substrate interactions in the CE4 family.Hide Abstract
Nareddy, P. K., Bobbili, K. B. & Swamy, M. J. (2017). International Journal of Biological Macromolecules, 95, 910-919.
A chitooligosaccharide-specific lectin has been purified from the phloem exudate of cucumber (Cucumis sativus) by affinity chromatography on chitin. The molecular weight of the cucumber phloem lectin (CPL) was determined as 51912.8 Da by mass spectrometry whereas SDS-PAGE yielded a single band with a subunit mass of 26 kDa, indicating that the protein is a homodimer. Peptide mass fingerprinting studies strongly suggest that CPL is identical to the 26 kDa phloem protein 2 (PP2) from cucumber. CD spectroscopy indicated that CPL is a predominantly β-sheets protein. Hemagglutination activity of CPL was mostly unaffected between 4 and 90°C and between pH 4.0 and 10.0, indicating functional stability of the protein. Isothermal titration calorimetric studies indicate that the CPL dimer binds to two chitooligosaccharide ((GlcNAc)2-6) molecules with association constants ranging from 1.0 × 103 to 17.5 × 105 M-1. The binding reaction was strongly enthalpy driven (δHb = −ve) with negative contribution from binding entropy (δSb = −ve). The enthalpy-driven nature of binding reactions suggests that hydrogen bonding and van der Waals interactions stabilize the CPL-chitooligosaccharide association. Enthalpy-entropy compensation was observed for the CPL-chitooligosaccharide interaction, indicating that water molecules play an important role in the binding process.Hide Abstract
Hamre, A. G., Schaupp, D., Eijsink, V. G. & Sørlie, M. (2015). FEBS Letters, 589(15), 1807-1812.
The enzymatic degradation of the closely related insoluble polysaccharides; cellulose (β(1–4)-linked glucose) by cellulases and chitin (β(1–4)-linked N-acetylglucosamine) by chitinases, is of large biological and economical importance. Processive enzymes with different inherent directionalities, i.e. attacking the polysaccharide chains from opposite ends, are crucial for the efficiency of this degradation process. While processive cellulases with complementary functions differ in structure and catalytic mechanism, processive chitinases belong to one single protein family with similar active site architectures. Using the unique model system of Serratia marcescens with two processive chitinases attacking opposite ends of the substrate, we here show that different directionalities of processivity are correlated to distinct differences in the kinetic signatures for hydrolysis of oligomeric tetra-N-acetyl chitotetraose.Hide Abstract
Wong, J. E. M. M., Midtgaard, S. R., Gysel, K., Thygesen, M. B., Sørensen, K. K., Jensen, K. J., Stougaard, J., Thirup, S. & Blaise, M. (2015). Acta Crystallographica Section D: Biological Crystallography, 71(3), 592-605.
LysM domains, which are frequently present as repetitive entities in both bacterial and plant proteins, are known to interact with carbohydrates containing N-acetylglucosamine (GlcNAc) moieties, such as chitin and peptidoglycan. In bacteria, the functional significance of the involvement of multiple LysM domains in substrate binding has so far lacked support from high-resolution structures of ligand-bound complexes. Here, a structural study of the Thermus thermophilus NlpC/P60 endopeptidase containing two LysM domains is presented. The crystal structure and small-angle X-ray scattering solution studies of this endopeptidase revealed the presence of a homodimer. The structure of the two LysM domains co-crystallized with N-acetyl-chitohexaose revealed a new intermolecular binding mode that may explain the differential interaction between LysM domains and short or long chitin oligomers. By combining the structural information with the three-dimensional model of peptidoglycan, a model suggesting how protein dimerization enhances the recognition of peptidoglycan is proposed.Hide Abstract
Hamre, A. G., Eide, K. B., Wold, H. H. & Sørlie, M. (2015). Carbohydrate Research, 407, 166-169.
For decades, the enzymatic conversion of recalcitrant polysaccharides such as cellulose and chitin was thought to solely rely on the synergistic action of hydrolytic enzymes, but recent work has shown that lytic polysaccharide monooxygenases (LPMOs) are important contributors to this process. Here, we have examined the initial rate enhancement an LPMO (CBP21) has on the hydrolytic enzymes (ChiA, ChiB, and ChiC) of the chitinolytic machinery of Serratia marcescens through determinations of apparent kcat (kcatapp) values on a β-chitin substrate. kcatapp values were determined to be
1.7±0.1 s-1 and 1.7±0.1 s-1 for the exo-active ChiA and ChiB, respectively and 1.2±0.1 s-1 for the endo-active ChiC. The addition of CBP21 boosted the kcatapp values of ChiA and ChiB giving values of 11.1±1.5 s-1 and 13.9±1.4 s-1, while there was no effect on ChiC (0.9±0.1 s-1).
Chang, Y. L., Liao, S. K. S., Chen, Y. C., Hung, W. T., Yu, H. M., Yang, W. B., Fang, J. M., Chen, C. H. & Lee, Y. C. (2011). Journal of mass spectrometry, 46(3), 247-255.
MALDI-MS provides a rapid and sensitive analysis of large biomolecules such as proteins and nucleic acids. However, oligo- and polysaccharides are less sensitive in MS analysis partly due to their neutral and hydrophilic nature to cause low ionization efficiency. In this study, four types of oligosaccharides including aldoses, aminoaldoses, alduronic acids and α-keto acids were modified by appropriate tags at the reducing termini to improve their ionization efficiency. Bradykinin (BK), a vasoactive nonapeptide (RPPGFSPFR), containing two arginine and two phenylalanine residues turned out to be an excellent MS signal enhancer for maltoheptaose, GlcNAc oligomers and oligogalacturonic acids. In the MALDI-TOF-MS analysis using 2,5-dihydroxybenzoic acid (2,5-DHB) as the matrix, the GalA4–BK and GalA5–BK conjugates prepared by reductive amination showed the detection limit at 0.1 fmol, i.e. ∼800-fold enhancement over the unmodified pentagalacturonic acids. The remarkable MS enhancement was attributable to the synergistic effect of the basic arginine residues for high proton affinity and the hydrophobic property phenylalanine residues for facile ionization. A tetrapeptide GFGR(OMe) and an arginine linked phenylenediamine (H2N)2Ph-R(OMe) were thus designed to act as potent tags of oligosaccharides in MS analysis. Interestingly, concurrent condensation and lactonization of α2,8-linked tetrasialic acid (SA4) was carried out with (H2N)2Ph-R(OMe) to obtain a quinoxalinone derivative, which showed > 200-fold enhancement over unmodified SA4 in the MALDI-TOF-MS analysis.Hide Abstract