Preparation of Defined Chitosan Oligosaccharides Using Chitin Deacetylases

Abstract

During the past decade, detailed studies using well-defined 'second generation' chitosans have amply proved that both their material properties and their biological activities are dependent on their molecular structure, in particular on their degree of polymerisation (DP) and their fraction of acetylation (F_A). Recent evidence suggests that the pattern of acetylation (PA), i.e., the sequence of acetylated and non-acetylated residues along the linear polymer, is equally important, but chitosan polymers with defined, non-random PA are not yet available. One way in which the PA will influence the bioactivities of chitosan polymers is their enzymatic degradation by sequence-dependent chitosan hydrolases present in the target tissues. The PA of the polymer substrates in conjunction with the subsite preferences of the hydrolases determine the type of oligomeric products and the kinetics of their production and further degradation. Thus, the bioactivities of chitosan polymers will at least in part be carried by the chitosan oligomers produced from them, possibly through their interaction with pattern recognition receptors in target cells. In contrast to polymers, partially acetylated chitosan oligosaccharides (paCOS) can be fully characterised concerning their DP, F_A, and PA, and chitin deacetylases (CDAs) with different and known regio-selectivities are currently emerging as efficient tools to produce fully defined paCOS in quantities sufficient to probe their bioactivities. In this review, we describe the current state of the art on how CDAs can be used in forward and reverse mode to produce all of the possible paCOS dimers, trimers, and tetramers, most of the pentamers and many of the hexamers. In addition, we describe the biotechnological production of the required fully acetylated and fully deacetylated oligomer substrates, as well as the purification and characterisation of the paCOS products.

Keywords: CDA; chitin; chitin deacetylase; chitinase; chito-oligosaccharide; chitosan; chitosanase; paCOS; pattern of acetylation; regio-selective.

Figure 1

Partially acetylated chito-oligosaccharides (paCOS) are characterised by three structural parameters, namely (i) the degree of polymerisation (DP), i.e., the number of monomeric units, (ii) the fraction of acetylation (F_A), i.e., the relative abundance of GlcNAc units, and (iii) the pattern of acetylation (PA), i.e., the sequence of GlcNAc and GlcN units.

Figure 2

Fungal chitin deacetylases (CDA) (right), which are better known than e.g., bacterial (left) or insect CDAs, typically possess four subsite binding sites (ranging from {–2} to {+1}) within a substrate binding cleft, highlighted in beige, each of which binds one monosaccharide subunit of the substrate, a chitin or chitosan oligomer or polymer. Each subsite can have its own specificity or preference for binding a N-acetylglucosamine or a glucosamine unit. The substrate binding cleft, shown in beige, may be delineated by six protein loops (L1 to L6) which may be flexible, allowing an induced fit of the enzyme. Left, schematic representation of the bacterial VcCDA from Vibrio for which the surrounding loops where first described; right, 3D-model of the fungal ClCDA from the Colletotrichum with bound substrate, chitin tetraose.

Figure 3

Chemo-enzymatic production of fully acetylated chitin oligomers and fully de-acetylated glucosamine oligomers. Fully acetylated chitin dimers A2 and trimers A3 can be generated from polymeric chitin by the action of a chitinase, fully de-acetylated dimers D2 and trimers D3 from polyglucosamine, itself prepared from chitin by sequential steps of alkaline de-acetylation, by the action of a chitosanase. Larger oligomers can be produced by the action of chitinase or chitosanase on partially acetylated chitosan polymers followed by alkaline de-acetylation yielding GlcN oligomers, or by chemical N-acetylation using acetic anhydride, yielding GlcNAc oligomers.

Figure 4

Fully acetylated chitin dimers A2, trimers A3, tetramers A4, and pentamers A5 can be produced biotechnologically in E. coli cell factories on kg scale. An E. coli strain expressing the chitin oligomer synthase NodC from Rhizobium yields A5. A strain expressing NodC together with NodB yields the α-mono-deacetylated pentamer DAAAA, which can be converted into A4 by GlcNase treatment to remove the deacetylated unit from the non-reducing end. A strain co-expressing NodC and a chitinase produces A3 and A2 which can be separated using chromatography.

Figure 5

Biotechnological preparation of the two dimeric paCOS DA and AD. DA can be prepared from chitobiose AA by the action of the CDA NodB from Rhizobium. AD can be prepared from chitobiose AA by the action of VcCDA from Vibrio. The substrate chitobiose AA can be prepared from polymeric chitin or from the chitin pentaose A5 or from the chitin tetraose A4 by the action of a (preferentially processive) chitinase such as SmChiB; the oligomeric substrates being produced by E. coli cell factories expressing the chitin oligomer synthase NodC from Rhizobium to yield A5 or NodC together with NodB to yield the α-mono-deacetylated pentamer DAAAA followed by GlcNase treatment to remove the deacetylated unit from the non-reducing end. In each case, the chitobiose has to be purified using liquid chromatography.

Figure 6

Biotechnological preparation of the six trimeric paCOS DAA, ADA, AAD, DDA, DAD, and ADD. DAA and ADA can be prepared from chitotriose AAA by the action of the CDA NodB from Rhizobium and VcCDA from Vibrio, respectively. DDA can then be produced by subsequent treatment with the other enzyme. Similarly, ADD and DAD can be prepared from DDD by the action of these two enzymes acting in reverse mode in the presence of excess amounts of acetate, and AAD can then be produced by subsequent reverse action of the other enzyme. The substrates for these reactions, AAA and DDD, can be prepared from polymeric chitin or polyglucosamine by the action of a chitinase or chitosanase, respectively. Alternatively, chitotriose can be produced by an E. coli cell factory strain expressing NodC from Rhizobium, yielding chitin pentaose which can then be cleaved, in vivo or in vitro, by a chitinase, yielding chitobiose and chitotriose. In each case, the trimeric product has to be purified using liquid chromatography.

Figure 7

Biotechnological preparation of the fourteen possible tetrameric paCOS using CDAs in forward or reverse mode on the fully acetylated or fully de-acetylated tetramers AAAA and DDDD, respectively, as described previously [34]. Both substrates can be prepared biotechnologically in good yields, AAAA by an E. coli cell factory expressing NodC and NodB to produce DAAAA followed by GlcNase treatment to yield AAAA, DDDD by digestion of polyglucosamine using an engineered chitosanase unable to cleave tetrameric substrates [70]. Both AAAA and DDDD need to be purified before converting them to paCOS using CDAs. Only five different CDAs are required, acting alone or in sequence, to produce all tetramers. For some of the tetramers, alternative routes exist and typically, chromatographic purification is required for the production of pattern-pure paCOS.

Figure 8

Experimentally proven and theoretical production routes for all thirty possible pentameric paCOS using known CDAs, alone or in combination, in forward or reverse mode. This overview shows the most feasible production route for each pentamer, but alternative routes exist for many of them. Since it has not yet been shown that PcCDA and BsPdaC also work in reverse mode, these hypothetical steps are marked with an asterisk.

Figure 9

Quantitative MS/MS sequencing of a mixture of isotopically labelled chitosan pentamers (A2D3). A partially acetylated chitosan polymer was enzymatically digested, the free amino groups of GlcN (D) residues within the oligomeric products obtained were acetylated using [²H₆]-acetic anhydride, resulting in [²H₃]N-acetylglucosamine (R), and their reducing ends were ¹⁸O-labelled. The labelled oligomers were separated using UHPLC hydrophilic interaction liquid chromatography (HILIC) and analyzed using ESI-MS/MS. The sequences (PA) of the chitosan oligomers in the triple-de-acetylated pentamers A2D3 was determined by using the relative abundances, shown in the boxes below the spectrum, of the fragment ions (denoted as B- and Y-ions according to nomenclature created by Domon and Costello in 1988 [74]), highlighted in brown.

Figure 10

Chromatographic separation of different paCOS. (A) SEC-chromatogram of the separation of a mixture of chitosans ranging from dimers to polymer according to their degree of polymerisation (DP). (B) Overlay-HILIC chromatogram of HILIC-purified tetrameric paCOS with different fractions of acetylation (F_A). (C) Overlay-HILIC-chromatogram of HILIC-purified mono-deacetylated tetrameric paCOS with different patterns of acetylation (PA).

Figure 10

Chromatographic separation of different paCOS. (A) SEC-chromatogram of the separation of a mixture of chitosans ranging from dimers to polymer according to their degree of polymerisation (DP). (B) Overlay-HILIC chromatogram of HILIC-purified tetrameric paCOS with different fractions of acetylation (F_A). (C) Overlay-HILIC-chromatogram of HILIC-purified mono-deacetylated tetrameric paCOS with different patterns of acetylation (PA).