Enzymatic production of all fourteen partially acetylated chitosan tetramers using different chitin deacetylases acting in forward or reverse mode

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.

Figure 1

Relative amounts of different chito-oligosaccharides after performing enzymatic N-acetylation of the chitosan tetramer (D4, [GlcN]₄) using different CDAs [BcCDA5, NodB, CvCDA, VcCDA, PesCDA, CnCDA2, PaCDA, CnCDA4, PgtCDA] for 98 h in 2 M sodium acetate buffer, pH 7. Data summarized in this figure have been generated by semi-quantitative HILIC-ESI-MS analysis and performed in triplicate; standard deviations are given by grey bars.

Figure 2

Base peak chromatograms of HILIC-ESI-MS analysis showing products after enzymatic deacetylation of the fully acetylated GlcNAc tetramer (A4, [GlcNAc]₄, filled circles) (A,C) and after enzymatic N-acetylation of the fully deacetylated GlcN tetramer (D4, [GlcN]_4, open circles) (B,D) using the bacterial CDAs NodB and VcCDA (A,B) and the fungal CDAs PesCDA, PaCDA and PgtCDA (C,D).

Figure 3

Mode of action of different CDAs (bacterial, viral, and fungal) on the chitin tetramer (A4, [GlcNAc]₄, filled circles) shown in the upper part, as well as on the chitosan tetramer (D4, [GlcN]_4, open circles) in the lower part. The reducing end of the (pa)COS always points to the right and is marked by a black circle. No activity is symbolized by an x, weak activity by an unfilled arrow, medium-strong activity by a grey-filled arrow, and strong activity by a black-filled arrow; arrows point to the unit of the (pa)COS which is preferentially de- or N-acetylated during either the 1^st, 2^nd, 3^rd, or 4^th attack by the different enzymes. Data summarized in this figure have been generated by (semi-)quantitative HILIC-ESI-MS analysis followed by ¹⁸O-labelling of the reducing end and MS/MS analysis.

Figure 4

Combination of bacterial CDAs during N-acetylation. (A) Base peak chromatogram of HILIC-ESI-MS analysis showing products after N-acetylation of the chitosan tetramer (D4, [GlcN]_4, open circles) by two bacterial CDAs (VcCDA, NodB) yielding in a first step the mono-acetylated paCOS by VcCDA, and in a second step the double-acetylated paCOS by NodB. (B) summarizes this production route.

Figure 5

Combination of bacterial and fungal CDAs during N-acetylation. (A) Base peak chromatograms of HILIC-ESI-MS analysis showing products after N-acetylation of the chitosan tetramer (D4, [GlcN]4, open circles) by several bacterial and fungal CDAs in different orders (CnCDA4, PesCDA, VcCDA, NodB), resulting in four different but new single-, double-, and triple-deacetylated products. Figure B summarizes these different production routes.

Figure 6

Production routes of all possible chitin and chitosan tetramers using CDAs to specifically deacetylate or N-acetylate (pa)COS; 1 represents the use of NodB, 2 of VcCDA, 3 of PesCDA, 4 of CnCDA4, and 5 of PgtCDA. Direction of routes are represented by arrows (grey: deacetylation, black: N-acetylation) and oligomers that are weak in color are not producible using this particular technique (de- or N-acetylation) (A) Production route of ten out of the 14 possible partially acetylated chitosan tetramers using different CDAs for deacetylation of the fully acetylated chitin tetramer as a substrate. (B) Production route of ten out of the 14 possible partially acetylated chitosan tetramers using different CDAs for N-acetylation under acetate-enriched conditions of the fully deacetylated chitosan tetramer as a substrate. (C) Overlay of production routes shown in part A (deacetylation) and B (N-acetylation), showing that all possible 14 partially acetylated chitosan tetramers are producible from the homotetramers A4 and D4.

Figure 7

Chromatographic purification up to analytical grade of the (pa)COS DDAD. (A) Base peak chromatogram of HILIC-ESI-MS analysis showing both buffer components (I, II, III, IV) and (pa)COS available in the sample after enzymatic N-acetylation of the GlcN tetramer by PesCDA and CnCDA4 (A3D1, A2D2, A1D3. (B) Base peak chromatogram of HILIC-ESI-MS analysis showing both buffer components (II) and (pa)COS available in the sample after purification over HILIC, rotational vacuum concentration and lyophilization.