TEMPO-mediated oxidation of chitin, regenerated chitin and N-acetylated chitosan

A commercial chitin, regenerated chitin prepared from chitin solutions in 6.8% NaOH and N-acetylated chitosans with degrees of N-acetylation (DNAc) of 77–93% were subjected to oxidization in water with NaClO and catalytic amounts of 2,2,6,6-tetramethylpiperidinyloxy radical (TEMPO) and NaBr. When regenerated chitin with DNAc of 87% and N-acetylated chitosan with DNAc of 93% were used as starting materials, water-soluble β-1,4-linked poly-N-acetylglucosaminuronic acid (chitouronic acid) Na salts with degrees of polymerization of ca. 300 were obtained quantitatively within 70 min. On the other hand, the original chitin and N-acetylated chitosan with DNAc of 77% did not give water-soluble products, owing to incomplete oxidation. The high crystallinity of the original chitin brought about low reactivity, and the high C2-amino group content of the N-acetylated chitosan with DNAc of 77% led to degradations rather than the selective oxidation at the C6 hydroxyls. The obtained chitouronic acid had low viscosities in water, and clear biodegradability by soil microorganisms.     

A new method for the quantification of chitin and chitosan in edible mushrooms

Along with β-glucans, chitin is the dominant component of the fungal cell wall. Chitosan, the deacetylated form of chitin, has found quite a number of biomedical and biotechnological applications recently. Mushroom chitin could be an important source for chitosan production. A direct determination of chitin and chitosan in mushrooms is of expedient interest. In this paper, a new method for the quantification of chitin and chitosan is described. This method is based on the specific reaction between polyiodide anions and chitosan and on measuring the optical density of the insoluble polyiodide–chitosan complex. After deacetylation, chitin can also be quantified. The specificity of the reaction is used to quantify the polymers in the presence of complex matrices. With this new spot assay, the chitin content of mycelia and fruiting bodies from several basidiomycetes and an ascomycete were analysed. The presented method could also be used for the determination in other samples as well. The chitin content of the analysed species varies between 0.4 and 9.8 g chitin per 100 g of dry mass. Chitosan could not be detected in our mushroom samples, indicating that the glucosamine units are mostly acetylated.     

Study on the production of chitin and chitosan from shrimp shell by using Bacillus subtilis fermentation

Fermentation of shrimp shell in jaggery broth using Bacillus subtilis for the production of chitin and chitosan was investigated. It was found that B. subtilis produced sufficient quantities of acid to remove the minerals from the shell and to prevent spoilage organisms. The protease enzyme in Bacillus species was responsible for the deprotenisation of the shell. The pH, proteolytic activity, extent of demineralization and deprotenisation were studied during fermentation. About 84% of the protein and 72% of the minerals were removed from the shrimp shell after fermentation. Mild acid and alkali treatments were given to produce characteristic chitin and their concentrations were standardized. Chitin was converted to chitosan by N-deacetylation and the properties of chitin and chitosan were studied. FTIR spectral analysis of chitin and chitosan prepared by the process was carried out and compared with spectra of commercially available samples.     

Biomaterials based on chitin and chitosan in wound dressing applications

Wound dressing is one of the most promising medical applications for chitin and chitosan. The adhesive nature of chitin and chitosan, together with their antifungal and bactericidal character, and their permeability to oxygen, is a very important property associated with the treatment of wounds and burns. Different derivatives of chitin and chitosan have been prepared for this purpose in the form of hydrogels, fibers, membranes, scaffolds and sponges. The purpose of this review is to take a closer look on the wound dressing applications of biomaterials based on chitin, chitosan and their derivatives in various forms in detail.     

Mining marine shellfish wastes for bioactive molecules: chitin and chitosan--Part A: extraction methods

Legal restrictions, high costs and environmental problems regarding the disposal of marine processing wastes have led to amplified interest in biotechnology research concerning the identification and extraction of additional high grade, low-volume by-products produced from shellfish waste treatments. Shellfish waste consisting of crustacean exoskeletons is currently the main source of biomass for chitin production. Chitin is a polysaccharide composed of N-acetyl-D-glucosamine units and the multidimensional utilization of chitin derivatives including chitosan, a deacetylated derivative of chitin, is due to a number of characteristics including: their polyelectrolyte and cationic nature, the presence of reactive groups, high adsorption capacities, bacteriostatic and fungistatic influences, making them very versatile biomolecules. Part A of this review aims to consolidate useful information concerning the methods used to extract and characterize chitin, chitosan and glucosamine obtained through industrial, microbial and enzymatic hydrolysis of shellfish waste.     

An easy,inexpensive, and sensitive method for the quantification of chitin in insect peritrophic membrane by image processing

ABSTRACT

Chitin, poly (β-(1→4)-N-acetyl-d-glucosamine), is an important biopolymer for insects that is utilized as a major component of peritrophic membrane. The chitin content in peritrophic membrane is of expedient interest from a pest control perspective, although it is hard to quantify chitin. In this study, we establish a facile method for the quantification of chitin in peritrophic membrane by image processing. In this method, chitin was indirectly quantified using chitosan–I₃^? complex, which exhibited a specific red-purple color. A calibration curve using a chitosan solution showed good linearity in a concentration range of 0.05–0.5 μg/μL. We quantified the amount of chitin in peritrophic membrane of Spodoptera litura (Lepidoptera: Noctuidae) larvae using this method. Throughout the study, only common inexpensive regents and easily attainable apparatuses were employed. This method can be easily applied to the sensitive quantification of the amounts of chitin and chitosan in materials by wide range of researchers.

Abbreviations: LOD: limit of detection; LOQ: limit of quantification; ROI: region of interest; RSD: relative standard deviation.     

Estimation of mycelial growth of basidiomycetes by means of chitin determination

After hydrolysis of chitin in 6 M HCl, the glucosamine produced was assayed colorimetrically. The pH of the hydrolysate was adjusted to a value close to three by addition of Na acetate; this procedure avoids the elimination of excess acid by evaporation under reduced pressure or freeze-drying. Under these conditions, the amount of glucosamine determined by the assay represented an average of 90% of the amount which would result from a total hydrolysis of the chitin. The method was used to assay the chitin in the mycelia of basidomycetes obtained in vitro. The measured amount of glucosamine was proportional to the mycelial biomass and allowed the estimation of fungal growth.     

Cloning and characterization of chsD, a chitin synthase-like gene of Aspergillus fumigatus

Abstract A chitin synthase-like gene ( chsD ) was isolated from an Aspergillus fumigatus genomic DNA library. Comparisons with the predicted amino acid sequence from chsD reveals low but significant similarity to chitin synthases, to other N acetylglucosaminyltransferases (NodC from Rhizopus spp., HasA from Streptococcus spp. and DG42 from vertebrates. A chsD⁻ mutant strain constructed by gene disruption has a 20% reduction in total mycelial chitin content; however, no differences between the wild-type strain and the chsD⁻ strain were found with respect to morphology, chitin synthase activity or virulence in a neutropenic murine model of aspergillosis. The results show that the chsD product has an important but inessential role in the synthesis of chitin in A. fumigatus .     

Determination of the degree of N-acetylation (DA) of chitin and chitosan in the presence of water by first derivative ATR FTIR spectroscopy

A new method for the determination of the degree of N-acetylation (DA) of chitin and chitosan is described using first derivative diamond ATR FTIR spectroscopy. Applying the derivative values of the amide III band at 1327 cm⁻¹ and the CH deformation band of the N-acetyl group at 1383 cm⁻¹ as measure of the N-acetyl content of the sample in relation to the derivative value of the bridge oxygen vibration at 1163 cm⁻¹ as internal standard, a linear correlation to the results of first derivative UV spectroscopy was obtained and confirmed by elemental analysis and Raman spectroscopy. The described method allows the determination of the degree of N-acetylation of chitosan and chitin in the presence of water thus making drying procedures unnecessary.     

Bioremediation of crude oil polluted seawater by a hydrocarbon-degrading bacterial strain immobilized on chitin and chitosan flakes

In this laboratory-scale study, we examined the potential of chitin and chitosan flakes obtained from shrimp wastes as carrier material for a hydrocarbon-degrading bacterial strain. Flakes decontamination, immobilization conditions and the survival of the immobilized bacterial strain under different storage temperatures were evaluated. The potential of immobilized hydrocarbon-degrading bacterial strain for crude oil polluted seawater bioremediation was tested in seawater microcosms. In terms of removal percentage of crude oil after 15 days, the microcosms treated with the immobilized inoculants proved to be the most successful. The inoculants formulated with chitin and chitosan as carrier materials improved the survival and the activity of the immobilized strain. It is important to emphasize that the inoculants formulated with chitin showed the best performance during storage and seawater bioremediation.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

Homogeneous synthesis and characterization of quaternized chitin in NaOH/urea aqueous solution

Water-soluble and white quaternized chitin (QC) was homogeneously synthesized by stirring transparent chitin solution (2%) in 8 wt%NaOH/4 wt% urea aqueous solution containing 2,3-Epoxypropyltrimethylammonium Chloride (EPTMAC) at 10 °C for 24 h. The structure and properties of quaternized chitin were characterized by FT-IR, XRD, ¹H NMR, GPC, element analysis and ζ-potential. The results indicate that quaternary groups were successfully incorporated onto chitin backbones and the degree of substitution (DS) of quaternary groups can be easily adjusted by changing the molar ratio of chitin unit to EPTMAC. Additionally, quaternized chitin shows better antibacterial activity against Escherichia coli and Staphylococcus aureus as compared with chitosan. Thus, this work provides a simply and “green” method to functionalize chitin and the resulting quaternized chitin may have potential applications in environmental, food and biomedical fields.     

Discovery of two new inhibitors of Botrytis cinerea chitin synthase by a chemical library screening

Chitin synthases polymerize UDP-GlcNAC to form chitin polymer, a key component of fungal cell wall biosynthesis. Furthermore, chitin synthases are desirable targets for fungicides since chitin is absent in plants and mammals. Two potent Botrytis cinerea chitin synthase inhibitors, 2,3,5-tri-O-benzyl-d-ribose (compound 1) and a 2,5-functionalized imidazole (compound 2) were identified by screening a chemical library. We adapted the wheat germ agglutinin (WGA) test for chitin synthase activity detection to allow miniaturization and robotization of the screen. Both identified compounds inhibited chitin synthases in vitro with IC₅₀ values of 1.8 and 10 μM, respectively. Compounds 1 and 2 were evaluated for their antifungal activity and were found to be active against B. cinerea BD90 strain with MIC values of 190 and 100 μM, respectively. Finally, we discovered that both compounds confer resistance to plant leaves against the attack of the fungus by reducing the propagation of lesions by 37% and 23%, respectively. Based on the inhibitory properties found in different assays, compounds 1 and 2 can be considered as antifungal hit inhibitors of chitin synthase, allowing further optimization of their pharmacological profile to improve their antifungal properties.     

Regulation of attachment, germination, and appressorium formation by zoospores ofLagenidium giganteum and related oomycetes by chitin, chitosan, and catecholamines

Summary Lagenidium giganteum (Oomycetes: Lagenidiales), a facultative parasite of mosquito larvae, infects the larval stage of most species of mosquitoes and a very limited number of alternate hosts. Host infection by this and other members of Oomycetes is initiated by motile, laterally biflagellate zoospores. Chemical bases for the various degrees of host specificity exhibited by these parasites is not known, but presumably involves receptors on the zoospore surface recognizing compounds either secreted by or on the surface of their hosts. Surface topography had no detectable effect onL. giganteum encystment or appressorium formation. Scanning electron microscopy documented the detachment of flagella during zoospore encystment. Bulbous knobs at the basal end of the detached flagellum were interpreted as encysting zoospores dropping the axoneme and/or the basal body and associated structures to which flagella are attached. Multiple signals appear to be involved in the initial steps ofL. giganteum host invasion. Zoospores of this parasite did not encyst on powdered preparations of chitin or chitosan (deacetylated chitin). Upon dissolution of chitosan in dilute acid followed by drying these solutions to form thin, transparent films, zoospores readily encysted. The degree of reacetylation of these films and the spacing of acetylated and deacetylated residues had no significant effect on zoospore encystment. Zoospores of a strain ofLagenidium myophilum isolated from marine shrimp, that also infects mosquito larvae, encysted on chitosan films. No encystment of spores of the plant parasitePhytophthora capsici was observed on chitin or chitosan films. Simulation of cuticle sclerotization by incubating chitosan films with different catecholamines and tyrosinase significantly reduced zoospore encystment. Zoospores that encysted on chitosan films did not germinate in distilled water. Germination could be induced by adding microgram quantities of bovine serum albumin or proteins secreted by motile zoospores into the water, and to a lesser degree by some amino acids, but not by various cations. Zoospores encysted and germinated on the pupal stage of some mosquito species. Appressoria were occasionally formed, but most subsequently sent out another mycelial branch, apparently without attempting to pierce the pupal cuticle. Methylation of pupal exuviae with ethereal diazomethane or methanol/HCl significantly increased zoospore encystment. Modification of chitin by catecholamines, lipids and protein on the epicuticular larval surface all affected host invasion.Abbreviations BSA bovine serum albumin - CID collision-induced dissociation - DOPA 3,4-dihydroxyphenylalanine - ESI-MS electrospray mass spectrometry - ESI-MS/MS tandem electrospray mass spectrometry - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - WGA wheat germ agglutinin - ZAP zoospore aggregation pheromone     

Chitosan but not chitin activates the inflammasome by a mechanism dependent upon phagocytosis

Chitin is an abundant polysaccharide found in fungal cell walls, crustacean shells, and insect exoskeletons. The immunological properties of both chitin and its deacetylated derivative chitosan are of relevance because of frequent natural exposure and their use in medical applications. Depending on the preparation studied and the end point measured, these compounds have been reported to induce allergic responses, inflammatory responses, or no response at all. We prepared highly purified chitosan and chitin and examined the capacity of these glycans to stimulate murine macrophages to release the inflammasome-associated cytokine IL-1β. We found that although chitosan was a potent NLRP3 inflammasome activator, acetylation of the chitosan to chitin resulted in a near total loss of activity. The size of the chitosan particles played an important role, with small particles eliciting the greatest activity. An inverse relationship between size and stimulatory activity was demonstrated using chitosan passed through size exclusion filters as well as with chitosan-coated beads of defined size. Partial digestion of chitosan with pepsin resulted in a larger fraction of small phagocytosable particles and more potent inflammasome activity. Inhibition of phagocytosis with cytochalasin D abolished the IL-1β stimulatory activity of chitosan, offering an explanation for why the largest particles were nearly devoid of activity. Thus, the deacetylated polysaccharide chitosan potently activates the NLRP3 inflammasome in a phagocytosis-dependent manner. In contrast, chitin is relatively inert.     

Optimization of the demineralization process for the extraction of chitin from Omani Portunidae segnis

Chitin is an organic polymer and it is the most frequent marine natural polysaccharide after cellulose. The main natural sources of chitin are exoskeletons of insects, mollusks, the cell walls of certain fungi and crustaceans such as crabs, shrimps and lobsters. The waste of these marine exoskeletons are pollutant for the environment, but these waste raw materials could be useful for production of commercial products like chitin. Chitin is an important raw material used for water treatment, agricultural, biomedical, biotechnological purposes, food and paper industry and cosmetics. Based on the variety of importance, the present targets of this study are to optimize the demineralization process for the removal of calcium and phosphate contents from the waste of Portunidae segnis (P. segnis) by using acid at ambient temperature and to characterize the isolated demineralized sample as well as the percentage of remaining calcium and phosphorus contents by using Inductively Coupled Plasma-Optical Emission Spectrometry (ICP-OES). The prepared waste carbs coarse powder samples of P. segnis were demineralized with seven different concentrations of hydrochloric acid at ambient temperature for 1 h. All the demineralization samples by the different concentrations were analyzed by using sensitive ICP-OES. The results based on ICP-OES showed that among the seven different concentrations used in the demineralization process for the isolation of chitin, the best was 2 M of HCl concentration for the production of chitin. The results also showed that the optimized concentration 2 M HCl gave the minimum concentration of calcium and phosphorus compared to other concentrations applied in this experiment. In conclusion, the optimized concentration for demineralization process could be used commercially for the isolation or commercial production of chitin for agricultural, biomedical and biotechnological purposes.     

Determination of the degree of deacetylation of chitin and chitosan by X-ray powder diffraction

A new method to determine the degree of deacetylation (DD) of alpha-chitin and chitosan in the range of 17-94% DD using X-ray powder diffraction (XRD) is proposed. The results were calibrated using (1)H NMR spectroscopy for chitosan and FTIR for chitin, in comparison with the potentiometric titration method. The results showed a good linear correlation between the CrI020 from XRD and the calibrated DD value. This method is found to be simple, rapid and nondestructive to the sample.     

Synthesis of chitin cycloalkyl ester derivatives and their physical properties

A series of acylated chitin derivatives was prepared by reacting chitin in a solution of trifluoroacetic anhydride and each of the cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl carboxylic acids. The degree of O-acyl substitution was in a range of 1.1-1.4 depending upon the nature of the cyclic acid added, as determined by FT-IR analysis. The solubility of the products in the organic solvents of DMF and THF increased with an increase in the cyclic chain length of the carboxylic acid. Thermal gravimetric analysis indicated that the products were stable up to 220 °C for chitin cyclopropanoate and cyclobutanoate, and 250 °C for chitin cyclopentanoate and cyclohexanoate. The surface morphology of the products by scanning electron microscopic analysis revealed porous and globular surface for chitin cyclobutanoate, cyclopentanoate, and cyclohexanoate, contrast to the dense and smooth organization for the cyclopropanoate.     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.