N-deacetylated Chitosan Research Articles

Effect of Chitosan Deacetylation on Its Affinity to Type III Collagen: A Molecular Dynamics Study.

The ability to form strong intermolecular interactions by linear glucosamine polysaccharides with collagen is strictly related to their nonlinear dynamic behavior and hence bio-lubricating features. Type III collagen plays a crucial role in tissue regeneration, and its presence in the articular cartilage affects its bio-technical features. In this study, the molecular dynamics methodology was applied to evaluate the effect of deacetylation degree on the chitosan affinity to type III collagen. The computational procedure employed docking and geometry optimizations of different chitosan structures characterized by randomly distributed deacetylated groups. The eight different degrees of deacetylation from 12.5% to 100% were taken into account. We found an increasing linear trend (R2 = 0.97) between deacetylation degree and the collagen–chitosan interaction energy. This can be explained by replacing weak hydrophobic contacts with more stable hydrogen bonds involving amino groups in N-deacetylated chitosan moieties. In this study, the properties of chitosan were compared with hyaluronic acid, which is a natural component of synovial fluid and cartilage. As we found, when the degree of deacetylation of chitosan was greater than 0.4, it exhibited a higher affinity for collagen than in the case of hyaluronic acid.

Diversity of family GH46 chitosanases in Kitasatospora setae KM-6054

The genome of Kitasatospora setae KM-6054, a soil actinomycete, has three genes encoding chitosanases belonging to GH46 family. The genes (csn1-3) were cloned in Streptomyces lividans and the corresponding enzymes were purified from the recombinant cultures. The csn2 clone yielded two proteins (Csn2BH and Csn2H) differing by the presence of a carbohydrate-binding domain. Sequence analysis showed that Csn1 and Csn2H were canonical GH46 chitosanases, while Csn3 resembled chitosanases from bacilli. The activity of the four chitosanases was tested in a variety of conditions and on diverse chitosan forms, including highly N-deacetylated chitosan or chitosan complexed with humic or polyphosphoric acid. Kinetic parameters were also determined. These tests unveiled the biochemical diversity among these chitosanases and the peculiarity of Csn3 compared with the other three enzymes. The observed biochemical diversity is discussed based on structural 3D models and sequence alignment. This is a first study of all the GH46 chitosanases produced by a single microbial strain.

Chiral stationary phases based on chitosan bis(4-methylphenylcarbamate)-(alkoxyformamide).

Highly N-deacetylated chitosan was chosen as a natural chiral origin for the synthesis of the selectors of chiral stationary phases. Therefore, chitosan was firstly acylated by various alkyl chloroformates yielding chitosan alkoxyformamides, and then these resulting products were further derivatized with 4-methylphenyl isocyanate to afford chitosan bis(4-methylphenylcarbamate)-(alkoxyformamide). A series of chiral stationary phases was prepared by coating these derivatives on 3-aminopropyl silica gel. The content of the derivatives on the chiral stationary phases was nearly 20% by weight. The chiral stationary phases prepared from chitosan bis(4-methylphenylcarbamate)-(ethoxyformamide) and chitosan bis(4-methylphenylcarbamate)-(isopropoxyformamide) comparatively showed better enantioseparation capability than those prepared from chitosan bis(4-methylphenylcarbamate)-(n-pentoxyformamide) and chitosan bis(4-methylphenylcarbamate)-(benzoxyformamide). The tolerance against organic solvents of the chiral stationary phase of chitosan bis(4-methylphenylcarbamate)-(ethoxyformamide) was investigated, and the results revealed that this phase can work in 100% ethyl acetate and 100% chloroform mobile phases. Because as-synthesized chiral selectors did not dissolve in many common organic solvents, the corresponding chiral stationary phases can be utilized in a wider range of mobile phases in comparison with conventional coating type chiral stationary phases of cellulose and amylose derivatives.

Chitooligosaccharide-2,5-anhydro-D-mannonic Acid

Chitooligosaccharide with one 2,5-anhydro-D-mannofuranose unit at the reducing end (COSamf) was prepared by nitrous deamination of fully N-deacetylated chitosan. The oxidation of the amf-unit by chlorite sodium was achieved in high yield. The chemical structure of the targeted chitooligosaccharide-2,5-anhydro-D-mannonic acid with a degree of polymerization ~10 was fully characterized by NMR spectroscopy, MALDI-TOF mass spectrometry and size-exclusion chromatography. The success of this synthesis opens the way to a new generation of end-functionalized COS building blocks.

4-(Hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-D-mannofuranose

Low molecular weight chitooligosaccharide with one 2,5-anhydro-D-mannofuranose unit at the reducing end (COSamf) was prepared by nitrous deamination of fully N-deacetylated chitosan. The functionalization of the amf unit by reductive amination with 4-(hexyloxy)aniline in presence of NaBH3CN was achieved in high yield. The chemical structure of the targeted 4-(hexyloxy)aniline-linked chitooligosaccharide-2,5-anhydro-D-mannofuranose was fully characterized by NMR spectroscopy, MALDI-TOF mass spectrometry and size-exclusion chromatography. This synthesis opens the way to a new generation of COSamf derivatives with potential amphiphilic properties.

Preparation and structure of chitosan soluble in wide pH range

Two kinds of chitosans, namely N-acetylated and N-deacetylated chitosan were prepared by the modified processes. They can dissolve in both acid and alkali solution. 13C NMR was used to study the basic solution of chitosan, and XRD, FT-IR and SEM were used to study the structure of N-acetylated and N-deacetylated chitosan. The result from X-ray diffraction showed that a transformation of crystal structure occurred during the N-acetylation or N-deacetylation process with the decrease of crystallinity and expansion of crystal lattices. FT-IR spectra revealed that the intermolecular and intramolecular hydrogen bonds were destroyed by both treatments and a looser structure was observed by the SEM. The lower crystallinity, the decreased intermolecular interactions, the more disordered and looser structure were easy for the permeation of LiOH/urea aqueous solution and coordinated with the breakage of intermolecular and intramolecular hydrogen bond by LiOH at low temperature, the prepared chitosans dissolved in LiOH/urea/H 2O mixture.

Chitosan-LiOH-urea aqueous solution—a novel water-based system for chitosan processing

A solution of partially N-deacetylated chitosan in aqueous lithium hydroxide (LiOH)/urea was prepared successfully through a freeze-thawing process and the dissolution behavior was studied. The results indicated that chitosan can directly dissolve in LiOH/urea aqueous solution. LiOH mainly contributed to the breakage of intramolecular and intermolecular hydrogen bonds in chitosan. Urea, LiOH, and chitosan formed inclusion compound (IC) with urea as the IC host, and the LiOH-chitosan complex as the guest. Aqueous 4.8 wt % LiOH/8.0 wt % urea was verified to be the optimal solvent for chitosan. The results of rheology and viscosity characterizations revealed that chitosan/4.8 wt % LiOH/8.0 wt % urea aqueous solution was pseudoplastic fluid, and was more stable than the solution of chitosan in acetic acid at ambient temperature.

Chemical Preparation and Structural Characterization of a Homogeneous Series of Chitin/Chitosan Oligomers

The preparation of a homogeneous series of chitin/chitosan oligomers (chito-oligomers) with the same distribution of degrees of polymerization (DP) ranging from 2 to 12, but with various average degrees of N-acetylation (DA) from 0 to 90% is described. This DA-series was obtained according to a two-step chemical process involving (i) the production of a well-defined mixture of glucosamine (GlcN) oligomers obtained by acid hydrolysis of a fully N-deacetylated chitosan and after selective precipitations of the hydrolysis products, and (ii) the partial N-acetylation of the GlcN units of these oligomers from a hydro-alcoholic solution of acetic anhydride in a controlled manner. The characterization of this series of samples with different DAs by proton nuclear magnetic resonance (1H NMR) spectroscopy and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) allowed us to determine their average DA and identify the main oligomer structures constituting each mixture. Furthermore, MALDI-TOF MS was particularly helpful to study the distribution evolution of the diverse oligomers as a function of DA for the main DPs from 3 to 7. The modeling of these distributions by means of a binomial law displayed that the chemical N-acetylation of low DP GlcN oligomers, produced in a homogeneous medium, occurs randomly along the oligosaccharide chains in accordance with a statistical (Bernoullian) arrangement. In this case, the relative proportion of each chito-oligomer present in the mixture can be estimated precisely as a function of DA considering oligomers of same DP.

Kinetics of hydrolysis of chitin/chitosan oligomers in concentrated hydrochloric acid

The kinetics of hydrolysis in concentrated hydrochloric acid (12.07 M) of the fully N-acetylated chitin tetramer (GlcNAc 4) and the fully N-deacetylated chitosan tetramer (GlcN 4) were followed by determining the amounts of the lower DP oligomers as a function of time. A theoretical model was developed to simulate the kinetics of hydrolysis of the three different glycosidic linkages in the tetramers. The model uses two different rate constants for the hydrolysis of the glycosidic bonds in the oligomers, assuming that the glycosidic bond next to one of the end residues are hydrolysed faster than the two other glycosidic linkages. The two rate constants were estimated by fitting model data to experimental results. The results show that the hydrolysis of the tetramers is a nonrandom process as the glycosidic bonds next to one of the end residues are hydrolysed 2.5 and 2.0 times faster as compared to the other glycosidic linkages in the fully N-acetylated and fully N-deacetylated tetramer, respectively. From previous results on other oligomers and the reaction mechanism, it is likely that the glycosidic bond that is hydrolysed fastest is the one next to the nonreducing end. The absolute values for the rate constants for the hydrolysis of the glycosidic linkages in GlcNAc 4 were found to be 50 times higher as compared to the glycosidic linkages in GlcN 4, due to the catalytic role of the N-acetyl group and the presence of the positively charged amino-group on the N-deacetylated sugar residue.

Evaluation of powder and tableting properties of chitosan

The aim of this study was to analyze the process of tablet formation and the properties of the resulting tablets for 3 N-deacetylated chitosans, with a degree of deacetylation of 80%, 85%, or 90%. Material properties, such as water content, particle size and morphology, glass transition temperature, and molecular weight were studied. The process of tablet formation was analyzed by 3-D modeling, Heckel analysis, the pressure time function, and energy calculations in combination with elastic recovery dependent on maximum relative density and time. The crushing force and the morphology of the final tablets were analyzed. Chitosans sorb twice as much water as microcrystalline cellulose (MCC), the particle size is comparable to Avicel PH 200, a special type of MCC, the particles look like shells, and the edges are bent. Molecular weight ranges from 80,000 to 210,000 kDa, the glass transition temperature (Tg) was not dependent on molecular weight. The chitosans deform ductilely as MCC; however, plastic deformation with regard to time and also pressure plasticity are higher than for MCC, especially for Chit 85, which has the lowest crystallinity and molecular weight. At high densification, fast elastic decompression is higher. 3-D modeling allowed the most precise analysis. Elastic recovery after tableting is higher than for MCC tablets and continues for some time after tableting. The crushing force of the resulting tablets is high owing to a reversible exceeding of Tg in the amorphous parts of the material. However, the crushing force is lower compared with MCC, since the crystallinity and the Tg of the chitosans are higher than for MCC. In summation, chitosans show plastic deformation during compression combined with high elasticity after tableting. Highly mechanically stable tablets result.

Properties of chitosanase from Bacillus cereus S1.

Chitosanase from Bacillus cereus S1 was purified, and the enzymatic properties were investigated. The molecular weight was estimated to 45,000 on SDS-PAGE. Optimum pH was about 6, and stable pH in the incubation at 40 degrees C for 60 min was 6-11. This chitosanase was stable in alkaline side. Optimum temperature was around 60 degrees C, and enzyme activity was relatively stable below 60 degrees C. The degradations of colloidal chitosan and carboxymethyl cellulose (CMC) were about 30 and 20% relative to the value of soluble chitosan, respectively, but colloidal chitin and crystalline cellulose were not almost hydrolyzed. On the other hand, S1 chitosanase adsorbed on colloidal chitin completely and by about 50% also on crystalline cellulose, in contrast to colloidal chitosan, which it did not adsorb. S1 chitosanase finally hydrolyzed 100% N-deacetylated chitosan (soluble state) to chitobiose (27.2%), chitotriose (40.6%), and chitotetraose (32.2%). In the hydrolysis of various chitooligosaccharides, chitobiose and chitotriose were not hydrolyzed, and chitotetraose was hydrolyzed to chitobiose. Chitobiose and chitotriose were released from chitopentaose and chitohexaose. From this specificity, it was hypothesized that the active site of S1 chitosanase recognized more than two glucosamine residues posited in both sides against splitting point for glucosamine polymer.

Studies on chitosan: 6. Relationship between N-acetyl group distribution pattern and chitinase digestibility of partially N-acetylated chitosans

The digestibility of partially N-acetylated chitosans by microbial chitinases was investigated in view of the distribution pattern of N-acetyl groups along the polysaccharide chain. Partially N-acetylated chitosans are classified into two groups; moderately N-deacetylated chitosans (MDC) with 10–30% acetyl content obtained by heterogeneous N-deacetylation of chitin and partially N-acetylated chitosans (PAC-H) with 20–70% acetyl content prepared by homogeneous N-acetylation of highly N-deacetylated chitosans (HDC). MDC have some blocks of N- acetyl- d- glucosamine (GlcNAc) sequences but PAC-H are random-type copolymers of GlcNAc and d-glucosamine. The apparent K m values of Streptomyces griseus chitinase were 0.14 g l −1 for 30% N-acetylated MDC and 0.16 g l −1 for 30% N-acetylated PAC-H. The K m values decreased with increased N-acetylation but the values for both MDC and PAC-H with similar acetyl content were almost the same. The chitinase from S. griseus could not distinguish the difference between block and random distributions of GlcNAc. The chitinases from Bacillus sp. and Bacillus sp. PI-7S also hydrolysed MDC and PAC-H in the same manner. From these results we conclude that sequences of GlcNAc are not necessary for recognition by these chitinases in contrast to lysozyme.

Studies on chitosan: 4. Lysozymic hydrolysis of partially N-acetylated chitosans

The lysozymic digestibility of partially N-acetylated chitosans was studied by measuring the reducing sugars produced and the molecular weights of their hydrolysates. Moderately N-deacetylated chitosans (MDC), obtained by N-deacetylation of chitin under heterogeneous conditions, were about four times more digestible at an early stage than partially N-acetylated chitosans (PAC-H) with similar acetyl content, prepared by N-acetylation of highly N-deacetylated chitosans under homogeneous conditions. The molecular weights of the hydrolysates of MDC decreased rapidly but gradually reached a constant value in contrast to the behaviour of PAC-H. The Km was 0.14 mM for 30% N-acetylated MDC and 0.12 mM for 65% N-acetylated PAC-H although the degree of N-acetylation of the latter was twice as much as the former. These differences were due to the different distribution patterns of N-acetyl groups in two types of the chitosans. MDC with 20-30% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues but PAC-H with about 30% acetyl content are random-type copolymers of N-acetyl-D-glucosamine and D-glucosamine units. PAC-H with more than 50% acetyl content have the sequences of more than three N-acetyl-D-glucosamine residues.

Determination of the degree of N-acetylation and the distribution of N-acetyl groups in partially N-deacetylated chitins (chitosans) by high-field n.m.r. spectroscopy

The composition and sequence of 2-acetamino-2-deoxy-β- d-glucose (GlcNAc) and 2-amino-2-deoxy-β- d-glucose (GlcN) residues in partially N-deacetylated chitosans, prepared under homogeneous and heterogeneous conditions, have been determined by 1H-n.m.r. spectroscopy. It was necessary to depolymerise the chitosan slightly by treatment with nitrous acid before spectroscopy. A sequence-dependent deshielding of H-1 of the GlcNAc residues made it possible to determine the proportions of the four possible diads. Chitosan prepared by N-deacetylation under homogenous conditions gave values for the diad frequencies that were roughly consistent with a random distribution of the N-acetyl groups. Samples prepared under heterogeneous conditions have a frequency of the GlcNAc-GlcNAc diad slightly higher than for a random (Bernoullian) distribution. The chitosans, prepared under both homogeneous and heterogeneous conditions, with a degre eof acetylation of 50% were soluble at neutral pH.

Studies on chitosan: 3. Evidence for the presence of random and block copolymer structures in partially N-acetylated chitosans

The chemical structures of moderately N-deacetylated chitosans (MDC) derived from chitin under heterogeneous reaction conditions and partially N-acetylated chitosans (PAC) derived from highly N-deacetylated chitosans (HDC) under homogeneous reaction conditions were deduced from the data of the stability of their solutions in alkaline media, the swelling behaviour and X-ray diffraction patterns of their films in connection with the degree of N-acetylation of them. The solutions of PAC with more than 51% acetyl content, which were prepared from HDC by N-acetylation, were stable and remained clear and homogeneous by adding 1.2 equivalents of NaOH. On the contrary the solutions of PAC with more than 52% acetyl content, which were prepared from MDC, became turbid by neutralization with less than 1.15 equivalents of NaOH. The films of PAC prepared from HDC were highly swollen in water. The degree of swelling of the chitosan film with 51% acetyl content, prepared from the 6% acetyl content chitosan, was 121% while that of the 53% acetyl content chitosan, prepared from the 30% acetyl content chitosan, was 28%. From these data it was possible to set up a hypothesis that PAC prepared from HDC were considered as random-type copolymers of N-acetyl-glucosamine and glucosamine units whereas MDC were considered as block-type copolymers.

Chitosan-elicited synthesis of callose and of coumarin derivatives in parsley cell suspension cultures

In suspension cultured cells of parsley (Petroselinum crispum), chitosan elicited a rapid deposition of the 1,3-ß-glucan callose on the cell wall and a slower formation of coumarins. With cells remaining in conditioned growth medium, fully N-deacetylated chitosans and partially N-acetylated chitosans were about equally active, the potency increased with the degree of polymerization up to several thousand and addition of reduced glutathione increased the sensitivity of the cells. These results indicate common initial events in the induction of callose and coumarin synthesis although two fully independent metabolic pathways are involved. When the cells were suspended in fresh growth medium, less chitosan was required, and fully N-deacetylated chitosan became the best callose elicitor.