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 monoacetylated 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 molecules.

ABSTRACTChitin is the second most abundant polysaccharide, present, e.g., in insect and arthropod exoskeletons and fungal cell walls. In some species or under specific conditions, chitin appears to be enzymatically de-N-acetylated to chitosan—e.g., when pathogenic fungi invade their host tissues. Here, the deacetylation of chitin is assumed to represent a pathogenicity mechanism protecting the fungus from the host's chitin-driven immune response. While highly specific chitin binding lectins are well known and easily available, this is not the case for chitosan-specific probes. This is partly due to the poor antigenicity of chitosan so that producing high-affinity, specific antibodies is difficult. Also, lectins with specificity to chitosan have been described but are not commercially available, and our attempts to reproduce the findings were not successful. We have, therefore, generated a fusion protein between a chitosanase inactivated by site-directed mutagenesis, the green fluorescent protein (GFP), and StrepII, as well as His6tags for purification and detection. The recombinant chitosan affinity protein (CAP) expressed inEscherichia coliwas shown to specifically bind to chitosan, but not to chitin, and the affinity increased with decreasing degree of acetylation.In vitro, CAP detection was possible either based on GFP fluorescence or using Strep-Tactin conjugates or anti-His5antibodies. CAP fluorescence microscopy revealed binding to the chitosan exposing endophytic infection structures of the wheat stem rust fungus, but not the chitin exposing ectophytic infection structures, verifying its suitability forin situchitosan staining.

Chitin is one of the most abundant biomolecules on earth, and its partially de-N-acetylated counterpart, chitosan, is one of the most promising biotechnological resources due to its diversity in structure and function. Recently, chitin and chitosan modifying enzymes (CCMEs) have gained increasing interest as tools to engineer chitosans with specific functions and reliable performance in biotechnological and biomedical applications. In a search for novel CCME, we isolated chitinolytic and chitosanolytic microorganisms from soils with more than ten-years history of chitin and chitosan exposure and screened them for chitinase and chitosanase isoenzymes as well as for their patterns of oligomeric products by incubating their secretomes with chitosan polymers. Of the 60 bacterial strains isolated, only eight were chitinolytic and/or chitosanolytic, while 20 out of 25 fungal isolates were chitinolytic and/or chitosanolytic. The bacterial isolates produced rather similar patterns of chitinolytic and chitosanolytic enzymes, while the fungal isolates produced a much broader range of different isoenzymes. Furthermore, diverse mixtures of oligosaccharides were formed when chitosan polymers were incubated with the secretomes of select fungal species. Our study indicates that soils with a history of chitin and chitosan exposure are a good source of novel CCME for chitosan bioengineering.

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 <i>β</i>-1,4-linked glucosamine (deacetylated/D) and <i>N</i>-acetylglucosamine (acetylated/A) units. To address this hypothesis, we prepared partially-acetylated chitosan oligosaccharides from a chitosan polymer (DA=35%, DP_w=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, <a>but also observed apparent DA-dependent priming activity, with the ADDD+DADD fraction proving remarkably effective</a>. 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 <i>bona fide</i> information-carrying molecules