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.

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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.

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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.

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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.

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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.

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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.

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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.

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Novel bi-modular GH19 chitinase with broad pH stability from a fibrolytic intestinal symbiont of Eisenia fetida, Cellulosimicrobium funkei HY-13.

Bai, L., Kim, J., Son, K. H., Chung, C. W., Shin, D. H., Ku, B. H., Kim, D. Y. & Park, H. Y. (2021). Biomolecules, 11(11), 1735.

Endo-type chitinase is the principal enzyme involved in the breakdown of N-acetyl-d-glucosamine-based oligomeric and polymeric materials through hydrolysis. The gene (966-bp) encoding a novel endo-type chitinase (ChiJ), which is comprised of an N-terminal chitin-binding domain type 3 and a C-terminal catalytic glycoside hydrolase family 19 domain, was identified from a fibrolytic intestinal symbiont of the earthworm Eisenia fetida, Cellulosimicrobium funkei HY-13. The highest endochitinase activity of the recombinant enzyme (rChiJ: 30.0 kDa) toward colloidal shrimp shell chitin was found at pH 5.5 and 55 °C and was considerably stable in a wide pH range (3.5–11.0). The enzyme exhibited the highest biocatalytic activity (338.8 U/mg) toward ethylene glycol chitin, preferentially degrading chitin polymers in the following order: ethylene glycol chitin > colloidal shrimp shell chitin > colloidal crab shell chitin. The enzymatic hydrolysis of N-acetyl-β-d-chitooligosaccharides with a degree of polymerization from two to six and colloidal shrimp shell chitin yielded primarily N,N-diacetyl-β-d-chitobiose together with a small amount of N-acetyl-d-glucosamine. The high chitin-degrading ability of inverting rChiJ with broad pH stability suggests that it can be exploited as a suitable biocatalyst for the preparation of N,N-diacetyl-β-d-chitobiose, which has been shown to alleviate metabolic dysfunction associated with type 2 diabetes.

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Heterologous expression and characterization of thermostable chitinase and β-N-acetylhexosaminidase from Caldicellulosiruptor acetigenus and their synergistic action on the bioconversion of chitin into N-acetyl-D-glucosamine.

Qin, X., Xin, Y., Su, X., Wang, X., Zhang, J., Tu, T., Wang, Y., Yao, B., Huang, H. & Luo, H. (2021). International Journal of Biological Macromolecules, 192, 250-257.

The bioconversion of chitin into N-acetyl-d-glucosamine (GlcNAc) using chitinolytic enzymes is one of the important avenues for chitin valorization. However, industrial applications of chitinolytic enzymes have been limited by their poor thermostability. Therefore, it is necessary to discover thermostable chitinolytic enzymes for GlcNAc production from chitin. In this study, two chitinolytic enzyme-encoding genes CaChiT and CaHex from Caldicellulosiruptor acetigenus were identified and heterologously expressed in Escherichia coli. The purified recombinant CaChiT and CaHex showed optimal activities at 70°C and 90°C respectively, and exhibited good thermostability over a range of temperature below 70°C and broad pH stability at pH range of 3.0-8.0. CaChiT and CaHex were active on colloidal chitin, pNP-(GlcNAc)2, pNP-(GlcNAc)3, and pNP-GlcNAc, pNP-(GlcNAc)2, pNP-(GlcNAc)3, pNP-Glc respectively. Besides, the chitin oligosaccharides and colloidal chitin hydrolysis profiles revealed that CaChiT degraded chitin chains through exo-mode of action. Furthermore, CaChiT and CaHex exhibited a synergistic effect in the degradation of colloidal chitin, reaching 0.60 mg/mL of GlcNAc production after 1 h incubation. These results suggested that a combination of CaChiT and CaHex have great potential for industrial applications in the enzymatic production of GlcNAc from chitin-containing biowastes.

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Biochemical characterization of a novel acidic chitinase with antifungal activity from Paenibacillus xylanexedens Z2-4.

Zhang, W., Ma, J., Yan, Q., Jiang, Z. & Yang, S. (2021). International Journal of Biological Macromolecules, 182, 1528-1536.

A chitinase gene (PxChi52) from Paenibacillus xylanexedens Z2-4 was cloned and heterologously expressed in Escherichia coli BL21 (DE3). PxChi52 shared the highest identity of 91% with a glycoside hydrolase family 18 chitinase (ChiD) from Bacillus circulans. The recombinant enzyme (PxChi52) was purified and biochemically characterized. PxChi52 had a molecular mass of 52.8 kDa. It was most active at pH 4.5 and 65°C, respectively, and stable in a wide pH range of 4.0-13.0 and up to 50°C. The enzyme exhibited the highest specific activity of 16.0 U/mg towards colloidal chitin, followed by ethylene glycol chitin (5.4 U/mg) and ball milled chitin (0.4 U/mg). The Km and Vmax values of PxChi52 towards colloidal chitin were determined to be 3.06 mg/mL and 71.38 U/mg, respectively, PxChi52 hydrolyzed colloidal chitin and chitooligosaccharides with degree of polymerization 2-5 to release mainly N-acetyl chitobiose. In addition, PxChi52 displayed inhibition effects on the growth of some phytopathogenic fungi, including Alternaria alstroemeriae, Botrytis cinerea, Rhizoctonia solani, Sclerotinia sclerotiorum and Valsa mali. The unique properties of PxChi52 may enable it potential application in agriculture field as a biocontrol agent.

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Chromatographic assays for the enzymatic degradation of chitin.

Mekasha, S., Tuveng, T. R., Vaaje-Kolstad, G. & Eijsink, V. G. (2021). Bio-protocol, 11(9), e4014.

Chitin is an insoluble linear polymer of β(1→4)-linked N-acetylglucosamine. Enzymatic cleavage of chitin chains can be achieved using hydrolytic enzymes, called chitinases, and/or oxidative enzymes, called lytic polysaccharide monooxygenases (LPMOs). These two groups of enzymes have different modes of action and yield different product types that require different analytical methods for detection and quantitation. While soluble chromogenic substrates are readily available for chitinases, proper insight into the activity of these enzymes can only be obtained by measuring activity toward their polymeric, insoluble substrate, chitin. For LPMOs, only assays using insoluble chitin are possible and relevant. Working with insoluble substrates complicates enzyme assays from substrate preparation to product analysis. Here, we describe typical set-ups for chitin degradation reactions and the chromatographic methods used for product analysis. Graphical abstract: Overview of chromatographic methods for assessing the enzymatic degradation of chitin.

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