Partially acetylated chitosan oligo- and polymers induce an oxidative burst in suspension cultured cells of the gymnosperm Araucaria angustifolia

International audienceSuspension-cultured cells were used to analyze the activation of defense responses in the conifer A. angustifolia, using as an elicitor purified chitosan polymers of different degrees of acetylation (DA 1−69%), chitin oligomers of different degrees of polymerization (DP 3−6), and chitosan oligomer of different DA (0−91%). Suspension cultured cells elicited with chitosan polymers reacted with a rapid and transient generation of H2O2, with chitosans of high DA (60 and 69%) being the most active ones. Chitosan oligomers of high DA (78 and 91%) induced substantial levels of H2O2, but fully acetylated chitin oligomers did not. When cultivated for 24−72 h in the presence of 1−10 μg mL−1 chitosan (DA 69%), cell cultures did not show alterations in the levels of enzymes related to defense responses, suggesting that, in A. angustifolia, the induction of an oxidative burst is not directly coupled to the induction of other defense reactions

Biactivity matrices for acetylated chitosan oligomers

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The Pattern of Acetylation Defines the Priming Activity of Chitosan Tetramers

The biological activity of chitosans depends on their degree of polymerization (DP) and degree of acetylation (DA). However, information could also be carried by the pattern of acetylation (PA): the sequence of β-1,4-linked glucosamine (deacetylated/D) and N-acetylglucosamine (acetylated/A) units. To address this hypothesis, we prepared partially-acetylated chitosan oligosaccharides from a chitosan polymer (DA=35%, DPw=905) using recombinant chitosan hydrolases with distinct substrate and cleavage specificities. The mixtures were separated into fractions DP4–DP12, which were tested for elicitor and priming activities in rice cells. We confirmed that both activities were influenced by DP, but also observed apparent DA-dependent priming activity, with the ADDD+DADD fraction proving remarkably effective. We then compared all four mono-acetylated tetramers prepared using different chitin deacetylases and observed significant differences in priming activity. This demonstrates for the first time that PA influences the biological activity of chitosans, which can now be recognized as bona fide information-carrying molecule

Enzymatic production of defined chitosan oligomers with a specific pattern of acetylation using a combination of chitin oligosaccharide deacetylases

Chitin and chitosan oligomers have diverse biological activities with potentially valuable applications in fields like medicine, cosmetics, or agriculture. These properties may depend not only on the degrees of polymerization and acetylation, but also on a specific pattern of acetylation (PA) that cannot be controlled when the oligomers are produced by chemical hydrolysis. To determine the influence of the PA on the biological activities, defined chitosan oligomers in sufficient amounts are needed. Chitosan oligomers with specific PA can be produced by enzymatic deacetylation of chitin oligomers, but the diversity is limited by the low number of chitin deacetylases available. We have produced specific chitosan oligomers which are deacetylated at the first two units starting from the non-reducing end by the combined use of two different chitin deacetylases, namely NodB from Rhizobium sp. GRH2 that deacetylates the first unit and COD from Vibrio cholerae that deacetylates the second unit starting from the non-reducing end. Both chitin deacetylases accept the product of each other resulting in production of chitosan oligomers with a novel and defined PA. When extended to further chitin deacetylases, this approach has the potential to yield a large range of novel chitosan oligomers with a fully defined architecture