Diacetyl-chitobiose

Content: 30 mg
Shipping Temperature: Ambient
Storage Temperature: Ambient
Physical Form: Powder
Stability: > 2 years under recommended storage conditions
CAS Number: 35061-50-8
Synonyms: di-N-acetylchitobiose, chitinbiose
Molecular Formula: C16H28N2O11
Molecular Weight: 424.4
Purity: > 95%
Substrate For (Enzyme): Chitobiase

High purity Diacetyl-chitobiose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.

Prepared from chitin.

We also offer other high purity oligosaccharides for research and analysis.

Documents
Certificate of Analysis
Safety Data Sheet
Data Sheet
Publications
Megazyme publication

Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.

Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.

Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.

Hide Abstract
Publication

NMR spectroscopic studies of chitin oligomers–Resolution of individual residues and characterization of minor amide cis conformations.

Wiesinger, P. & Nestor, G. (2025). Carbohydrate Polymers, VO. 351, 123122.

Chitin is the second most abundant biopolymer in nature after cellulose and is composed of N-acetylglucosamine (GlcNAc) connected via β(1 → 4)-glycosidic bonds. Despite its prominence in nature and diverse roles in pharmaceutical and food technological applications, there is still a need to develop methods to study structure and function of chitin and its corresponding oligomers. Efforts have been made to analyse chitin oligomers by NMR spectroscopy, but spectral overlap has prevented any differentiation between the interior residues. In this study, chitin oligomers up to hexaose with natural abundance of 15N were analysed with NMR spectroscopy in aqueous solution. Different 1H,15N-HSQC pulse sequences were evaluated to obtain the best resolution and sensitivity. Interior residues were resolved in the 15N dimension and detailed chemical shifts of amide proton and nitrogen are reported for the first time. Additionally, all oligomers were analysed for the presence of the amide cis form and its corresponding chemical shifts were assigned. This study exploits the information that can be obtained from chitin oligomers with NMR spectroscopy and depicts methods for detailed analysis of glycans containing oligomers of N-acetylglucosamine.

Hide Abstract
Publication

Exploring roles of the chitinase ChiC in modulating Pseudomonas aeruginosa virulence phenotypes.

Edvardsen, P. K. T., Askarian, F., Zurich, R., Nizet, V. & Vaaje-Kolstad, G. (2024). Microbiology Spectrum, 12(7), e00546-24.

Chitinases are ubiquitous enzymes involved in biomass degradation and chitin turnover in nature. Pseudomonas aeruginosa (PA), an opportunistic human pathogen, expresses ChiC, a secreted glycoside hydrolase 18 family chitinase. Despite speculation about ChiC's role in PA disease pathogenesis, there is scant evidence supporting this hypothesis. Since PA cannot catabolize chitin, we investigated the potential function(s) of ChiC in PA pathophysiology. Our findings show that ChiC exhibits activity against both insoluble (α- and β-chitin) and soluble chitooligosaccharides. Enzyme kinetics toward (GlcNAc)4 revealed a kcat of 6.50 s-1 and a KM of 1.38 mM, the latter remarkably high for a canonical chitinase. In our label-free proteomics investigation, ChiC was among the most abundant proteins in the Pel biofilm, suggesting a potential contribution to PA biofilm formation. Using an intratracheal challenge model of PA pneumonia, the chiC::ISphoA/hah transposon insertion mutant paradoxically showed slightly increased virulence compared to the wild-type parent strain. Our results indicate that ChiC is a genuine chitinase that contributes to a PA pathoadaptive pathway.IMPORTANCEIn addition to performing chitin degradation, chitinases from the glycoside hydrolase 18 family have been found to play important roles during pathogenic bacterial infection. Pseudomonas aeruginosa is an opportunistic pathogen capable of causing pneumonia in immunocompromised individuals. Despite not being able to grow on chitin, the bacterium produces a chitinase (ChiC) with hitherto unknown function. This study describes an in-depth characterization of ChiC, focusing on its potential contribution to the bacterium's disease-causing ability. We demonstrate that ChiC can degrade both polymeric chitin and chitooligosaccharides, and proteomic analysis of Pseudomonas aeruginosa biofilm revealed an abundance of ChiC, hinting at a potential role in biofilm formation. Surprisingly, a mutant strain incapable of ChiC production showed higher virulence than the wild-type strain. While ChiC appears to be a genuine chitinase, further investigation is required to fully elucidate its contribution to Pseudomonas aeruginosa virulence, an important task given the evident health risk posed by this bacterium.

Hide Abstract
Publication

Mutational dissection of a hole hopping route in a lytic polysaccharide monooxygenase (LPMO). 

Ayuso-Fernández, I., Emrich-Mills, T. Z., Haak, J., Golten, O., Hall, K. R., Schwaiger, L., Moe, T. S., Stepnov, A. A., Ludwig, R., Cutsail III, G. E., Sorlie, M., Rohr, A. K. & Eijsink, V. G. (2024). Nature Communications, 15(1), 3975.

Oxidoreductases have evolved tyrosine/tryptophan pathways that channel highly oxidizing holes away from the active site to avoid damage. Here we dissect such a pathway in a bacterial LPMO, member of a widespread family of C-H bond activating enzymes with outstanding industrial potential. We show that a strictly conserved tryptophan is critical for radical formation and hole transference and that holes traverse the protein to reach a tyrosine-histidine pair in the protein’s surface. Real-time monitoring of radical formation reveals a clear correlation between the efficiency of hole transference and enzyme performance under oxidative stress. Residues involved in this pathway vary considerably between natural LPMOs, which could reflect adaptation to different ecological niches. Importantly, we show that enzyme activity is increased in a variant with slower radical transference, providing experimental evidence for a previously postulated trade-off between activity and redox robustness.

Hide Abstract
Publication

Fast insights into chitosan-cleaving enzymes by simultaneous analysis of polymers and oligomers through size exclusion chromatography.

Hellmann, M. J., Moerschbacher, B. M. & Cord-Landwehr, S. (2024). Scientific Reports, 14(1), 3417.

The thorough characterization of chitosan-cleaving enzymes is crucial to unveil structure–function relationships of this promising class of biomolecules for both, enzymatic fingerprinting analyses and to use the enzymes as biotechnological tools to produce tailor-made chitosans for diverse applications. Analyzing polymeric substrates as well as oligomeric products has been established as an effective way to understand the actions of enzymes, but it currently requires separate, rather laborious methods to obtain the full picture. Here, we present ultra high performance size exclusion chromatography coupled to refractive index and mass spectrometry detection (UHPSEC-RI-MS) as a straightforward method for the semi-quantitative analysis of chitosan oligomers of up to ten monomers in length. Additionally, the method allows to determine the average molecular weight of the remaining polymers and its distribution. By sampling live from an ongoing enzymatic reaction, UHPSEC-RI-MS offers the unique opportunity to analyze polymers and oligomers simultaneously—i.e., to monitor the molecular weight reduction of the polymeric substrate over the course of the digestion, while at the same time analyzing the emerging oligomeric products in a semi-quantitative manner. In this way, a single simple analysis yields detailed insights into an enzyme’s action on a given substrate.

Hide Abstract
Publication

Candida albicans chitinase 3 with potential as a vaccine antigen: production, purification, and characterisation.

Costa‐Barbosa, A., Ferreira, D., Pacheco, M. I., Casal, M., Duarte, H. O., Gomes, C., Barbosa, A. M., Torrado, E., Sampaio, P. & Collins, T. (2023). Biotechnology Journal, 2300219.

Chitinases are widely studied enzymes that have already found widespread application. Their continued development and valorisation will be driven by the identification of new and improved variants and/or novel applications bringing benefits to industry and society. We previously identified a novel application for chitinases wherein the Candida albicans cell wall surface chitinase 3 (Cht3) was shown to have potential in vaccine applications as a subunit antigen against fungal infections. In the present study, this enzyme was investigated further, developing production and purification protocols, enriching our understanding of its properties, and advancing its application potential. Cht3 was heterologously expressed in Pichia pastoris and a 4-step purification protocol developed and optimised: this involves activated carbon treatment, hydrophobic interaction chromatography, ammonium sulphate precipitation, and gel filtration chromatography. The recombinant enzyme was shown to be mainly O-glycosylated and to retain the epitopes of the native protein. Functional studies showed it to be highly specific, displaying activity on chitin, chitosan, and chito-oligosaccharides larger than chitotriose only. Furthermore, it was shown to be a stable enzyme, exhibiting activity, and stability over broad pH and temperature ranges. This study represents an important step forward in our understanding of Cht3 and contributes to its development for application.

Hide Abstract
Publication

The Ustilago maydis AA10 LPMO is active on fungal cell wall chitin.

Yao, R. A., Reyre, J. L., Tamburrini, K. C., Haon, M., Tranquet, O., Nalubothula, A., Mukherjee, S., Gall, S. L., Grisel, S., Longhi, S., Madhuprakash, J., Bissaro, B. & Berrin, J. G. (2023). Applied and Environmental Microbiology, 89(10), e00573-23.

Lytic polysaccharide monooxygenases (LPMOs) can perform oxidative cleavage of glycosidic bonds in carbohydrate polymers (e.g., cellulose, chitin), making them more accessible to hydrolytic enzymes. While most studies have so far mainly explored the role of LPMOs in a (plant) biomass conversion context, alternative roles and paradigms begin to emerge. The AA10 LPMOs are active on chitin and/or cellulose and mostly found in bacteria and in some viruses and archaea. Interestingly, AA10-encoding genes are also encountered in some pathogenic fungi of the Ustilaginomycetes class, such as Ustilago maydis, responsible for corn smut disease. Transcriptomic studies have shown the overexpression of the AA10 gene during the infectious cycle of U. maydis. In fact, U. maydis has a unique AA10 gene that codes for a catalytic domain appended with a C-terminal disordered region. To date, there is no public report on fungal AA10 LPMOs. In this study, we successfully produced the catalytic domain of this LPMO (UmAA10_cd) in Pichia pastoris and carried out its biochemical characterization. Our results show that UmAA10_cd oxidatively cleaves α- and β-chitin with C1 regioselectivity and boosts chitin hydrolysis by a GH18 chitinase from U. maydis (UmGH18A). Using a biologically relevant substrate, we show that UmAA10_cd exhibits enzymatic activity on U. maydis fungal cell wall chitin and promotes its hydrolysis by UmGH18A. These results represent an important step toward the understanding of the role of LPMOs in the fungal cell wall remodeling process during the fungal life cycle.

Hide Abstract
Publication

Structural characterization of ligand binding and pH-specific enzymatic activity of mouse Acidic Mammalian Chitinase.

Díaz, R. E., Ecker, A. K., Correy, G. J., Asthana, P., Young, I. D., Faust, B., Thompson, M. C., Seiple, I. B., Van Dyken, S., Locksley, R. M. & Fraser, J. S. (2023). bioRxiv, 2023-06.

Chitin is an abundant biopolymer and pathogen-associated molecular pattern that stimulates a host innate immune response. Mammals express chitin-binding and chitin-degrading proteins to remove chitin from the body. One of these proteins, Acidic Mammalian Chitinase (AMCase), is an enzyme known for its ability to function under acidic conditions in the stomach but is also active in tissues with more neutral pHs, such as the lung. Here, we used a combination of biochemical, structural, and computational modeling approaches to examine how the mouse homolog (mAMCase) can act in both acidic and neutral environments. We measured kinetic properties of mAMCase activity across a broad pH range, quantifying its unusual dual activity optima at pH 2 and 7. We also solved high resolution crystal structures of mAMCase in complex with chitin, where we identified extensive conformational ligand heterogeneity. Leveraging these data, we conducted molecular dynamics simulations that suggest how a key catalytic residue could be protonated via distinct mechanisms in each of the two environmental pH ranges. These results integrate structural, biochemical, and computational approaches to deliver a more complete understanding of the catalytic mechanism governing mAMCase activity at different pH. Engineering proteins with tunable pH optima may provide new opportunities to develop improved enzyme variants, including AMCase, for therapeutic purposes in chitin degradation.

Hide Abstract
Publication

Family 92 carbohydrate-binding modules specific for β-1, 6-glucans increase the thermostability of a bacterial chitinase.

Li, H., Lu, Z., Hao, M. S., Kvammen, A., Inman, A. R., Srivastava, V., Bulone, V. & McKee, L. S. (2023). Biochimie, 212, 153-160.

In biomass-processing industries there is a need for enzymes that can withstand high temperatures. Extensive research efforts have been dedicated to finding new thermostable enzymes as well as developing new means of stabilising existing enzymes. The attachment of a stable non-catalytic domain to an enzyme can, in some instances, protect a biocatalyst from thermal denaturation. Carbohydrate-binding modules (CBMs) are non-catalytic domains typically found appended to biomass-degrading or modifying enzymes, such as glycoside hydrolases (GHs). Most often, CBMs interact with the same polysaccharide as their enzyme partners, leading to an enhanced reaction rate via the promotion of enzyme-substrate interactions. Contradictory to this general concept, we show an example of a chitin-degrading enzyme from GH family 18 that is appended to two CBM domains from family 92, both of which bind preferentially to the non-substrate polysaccharide β-1,6-glucan. During chitin hydrolysis, the CBMs do not contribute to enzyme-substrate interactions but instead confer a 10-15°C increase in enzyme thermal stability. We propose that CBM92 domains may have a natural enzyme stabilisation role in some cases, which may be relevant to enzyme design for high-temperature applications in biorefinery.

Hide Abstract
Publication

Reductants fuel lytic polysaccharide monooxygenase activity in a pH‐dependent manner.

Golten, O., Ayuso‐Fernández, I., Hall, K. R., Stepnov, A. A., Sørlie, M., Røhr, Å. K. & Eijsink, V. G. (2023). FEBS letters, 597(10), 1363-1374.

Polysaccharide-degrading mono-copper lytic polysaccharide monooxygenases (LPMOs) are efficient peroxygenases that require electron donors (reductants) to remain in the active Cu(I) form and to generate the H2O2 co-substrate from molecular oxygen. Here, we show how commonly used reductants affect LPMO catalysis in a pH-dependent manner. Between pH 6.0 and 8.0, reactions with ascorbic acid show little pH dependency, whereas reactions with gallic acid become much faster at increased pH. These dependencies correlate with the reductant ionization state, which affects its ability to react with molecular oxygen and generate H2O2. The correlation does not apply to l-cysteine because, as shown by stopped-flow kinetics, increased H2O2 production at higher pH is counteracted by increased binding of l-cysteine to the copper active site. The findings highlight the importance of the choice of reductant and pH in LPMO reactions.

Hide Abstract
Safety Information
Symbol : Not Applicable
Signal Word : Not Applicable
Hazard Statements : Not Applicable
Precautionary Statements : Not Applicable
Safety Data Sheet
Customers also viewed
Tetraacetyl-chitotetraose O-CHI4
Tetraacetyl-chitotetraose
1,4-beta-D-N-Acetylglucosaminyl-D-Glucosamine O-CHIN2A
1,4-β-D-N-Acetylglucosaminyl-D-Glucosamine