Abstract
Some of the most abundant biomolecules on earth are the polysaccharides chitin and chitosan of which especially the oligomeric fractions have been extensively studied regarding their biological activities. However, most of these studies have not been able to assess the activity of a single, defined, partially acetylated chitosan oligosaccharide (paCOS). Instead, they have typically analyzed chemically produced, rather poorly characterized mixtures, at best with a single, defined degree of polymerization (DP) and a known average degree of acetylation (DA), as no pure and well-defined paCOS are currently available. We here present data on the enzymatic production of all 14 possible partially acetylated chitosan tetramers, out of which four were purified (>95%) regarding DP, DA, and pattern of acetylation (PA). We used bacterial, fungal, and viral chitin deacetylases (CDAs), either to partially deacetylate the chitin tetramer; or to partially re-N-acetylate the glucosamine tetramer. Both reactions proceeded with surprisingly strong and enzyme-specific regio-specificity. These pure and fully defined chitosans will allow to investigate the particular influence of DP, DA, and PA on the biological activities of chitosans, improving our basic understanding of their modes of action, e.g. their molecular perception by patter recognition receptors, but also increasing their usefulness in industrial applications.
Highlights
As people and industries become more concerned with finding sustainable, natural solutions to modern issues, the use of natural compounds, such as the polysaccharides chitin and chitosan, have skyrocketed in various industries, in agriculture and medicine
Though experimental evidence for the hypothesis is missing owing to a lack of the required isomeric chitosan octamers, inhibitor activity is thought to depend on the alternating pattern of acetylation (PA) of the partially acetylated chitosans (paCOS) used which possesses one face with four exposed, hydrophobic acetyl groups able to bind the receptor, but which exposes four charged free amino groups on the opposite face, preventing binding of a second receptor molecule
GRH2, VcCDA from Vibrio cholerae, BcCDA5 from Bacillus licheniformis), five fungal chitin deacetylases (CDAs) (CnCDA2 and CnCDA4 from Cryptococcus neoformans, PesCDA from Pestalotiopsis spec., PgtCDA from Puccinia graminis f. sp. tritici, PaCDA from Podospora anserina) as well as one viral CDA (CvCDA from Chlorovirus CVK2), which were all heterologously expressed in either E. coli or H. polymorpha and purified using affinity chromatography
Summary
As people and industries become more concerned with finding sustainable, natural solutions to modern issues, the use of natural compounds, such as the polysaccharides chitin and chitosan, have skyrocketed in various industries, in agriculture and medicine. When taking a closer look at most studies published dealing with the biological activities of COS and, paCOS, very few have used pure and well-defined oligomers; instead, broad mixtures have most often been used These mixtures are typically produced by partial chemical or enzymatic depolymerisation of chitin or chitosan polymers which themselves are often only poorly characterised[17,18,19]. The pattern of acetylation (PA) —another characteristic of chitosans believed to strongly influence biological activities13,22,23 — is almost entirely unexplored, mostly because there are no known methods to separate isomeric paCOS, i.e. paCOS with identical DP and DA, but different PAs, and because protocols for the organic synthesis of defined paCOS are still in their infancies[21,24] Using such mixtures when investigating biological activities of chitins and chitosans will likely cause the results of such studies to vary and not be adequately reproducible, as some of the oligomers in the mixtures may have a certain biological effect while others do not. Fully defined paCOS are urgently required to further our understanding of structure-function relationships of the biological activities of partially acetylated chitosans
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