Chitin and derivative chitosan-based structures - Preparation strategies aided by deep eutectic solvents: A review.

The high molecular weight of chitin, as a biopolymer, challenges its extraction due to its insolubility in the solvents. Also, chitosan, as the N-deacetylated form of chitin, can be employed as a primary material for different industries. The low mechanical stability and poor plasticity of chitosan films, as a result of incompatible interaction between chitosan and the used solvent, have limited its industrialization. Deep eutectic solvents (DESs), as novel solvents, can solve the extraction difficulties of chitin, and the low mechanical stability and weak plasticity of chitosan films. Also, DESs can be considered for the different chitosan and chitin productions, including chitin nanocrystal and nanofiber, N,N,N-trimethyl-chitosan, chitosan-based imprinted structures, and DES-chitosan-based beads and monoliths. This review aims to focus on the preparation and characterization (chemistry and morphology) of DES-chitin-based and DES-chitosan-based structures to understand the influence of the incorporation of DESs into the chitin and chitosan structure.

Synthesis of mixed chitin esters with long fatty and bulky acyl substituents in ionic liquid.

This study revealed that mixed chitin esters with long fatty and bulky acyl substituents were efficiently synthesized by acylation using acyl chlorides in the presence of pyridine and N,N-dimethyl-4-aminopyridine in an ionic liquid, 1-allyl-3-methylimidazolium bromide (AMIMBr), at 100 °C for 24 h. A stearoyl group was selected as the first substituent, which was combined with different long fatty and bulky acyl groups as the second substituents. In addition to IR analysis of the products, which suggested progress of the acylation, 1H NMR measurement was allowed for structural confirmation for high degrees of substitution (DSs) of the desired derivatives in CDCl3/CF3CO2H solvents. Crystalline structures and thermal property of the products were evaluated by powder X-ray diffraction and differential scanning calorimetry measurements, respectively. All the products showed film formability by casting from solutions in chloroform or chloroform/trifluoroacetic acid solvents. The occurrence of halogen exchange between acyl chlorides and AMIMBr in the present system was speculated to produce highly reactive acyl bromides in situ, which efficiently reacted with hydroxy groups in chitin to obtain high DS products.

Functionalisation of the non-reducing end of chitin by selective periodate oxidation: A new approach to form complex block polysaccharides and water-soluble chitin-based block polymers.

Most polysaccharides used in polysaccharide-based block copolymers are attached to the second block through the reducing end, due to the few and highly polysaccharide specific non-reducing end (NRE) functionalisation methods available. Chitin oligomers, prepared by nitrous acid degradation of chitosan (AnM) can, however, be selectively oxidised by periodate since they only possess a single vicinal diol in the NRE residue. Here, we show that both aldehydes formed after oxidation are highly reactive towards bifunctional oxyamines and hydrazide linkers. Sub-stochiometric amounts of linkers resulted in conjugation of AnM oligomers through both chain termini to yield a discrete distribution of 'polymerised' oligomers. Such chitin-based block polymers were, in contrast to chitins of the same chain lengths, water-soluble. Oxidised AnM oligomers, functionalised at both termini can also enable the preparation of more complex block polysaccharides such as ABA- or ABC-type.

Systematic Structural Characterization of Chitooligosaccharides Enabled by Automated Glycan Assembly.

Chitin, a polymer composed of ß(1-4)-linked N-acetyl-glucosamine monomers, and its partially deacetylated analogue chitosan, are abundant biopolymers with outstanding mechanical as well as elastic properties. Their degradation products, chitooligosaccharides (COS), can trigger the innate immune response in humans and plants. Both material and biological properties are dependent on polymer length, acetylation, as well as the pH. Without well-defined samples, a complete molecular description of these factors is still missing. Automated glycan assembly (AGA) enabled rapid access to synthetic well-defined COS. Chitin-cellulose hybrid oligomers were prepared as important tools for a systematic structural analysis. Intramolecular interactions, identified by molecular dynamics simulations and NMR analysis, underscore the importance of the chitosan amino group for the stabilization of specific geometries.

Synthesis and biophysical analysis of Naringin-Chitooligosaccharide complex.

In this study, new complexes of Naringin and Chitooligosaccharide (Nari-COS) at different mole ratios (1:1, 1:5, 1:10) were prepared by spray-drying method so as to enhance the water solubility and weaken the bitterness of naringin. At the same time, the antioxidant and the antibacterial properties of this complex were evaluated. SEM, FTIR, 1H NMR analysis confirmed the successful synthesis of Nari-COS formed through hydrogen bonds between the A, B rings of naringin and COS. Nari-COS exhibited significantly better water solubility, reduced bitterness, stronger antioxidant capacity, and enhanced antibacterial property in comparison to pure naringin, benefitting the extensive application of natural products in foods.

Effect of the Degree of Acetylation of Chitin Nonwoven Fabrics for Promoting Wound Healing.

Chitin and chitosan have been extensively used as wound dressings because of their special functions to promote wound healing. However, there was little focus on the effects of the degree of acetylation (DA) on wound healing. In this work, the regenerated chitin nonwoven fabrics with DA values of 90, 71, 60, and 42% were prepared, and the morphology and physical performances of the fabrics were characterized. Moreover, the effects of DA of the chitin nonwoven fabrics on wound recovery were studied with a full-thickness skin defect model in rats. In vitro experiments indicated that the chitin nonwoven fabrics exhibited good biocompatibility and blood compatibility and a low blood-clotting index (BCI). In vivo experiments revealed that the chitin nonwoven fabrics could accelerate wound healing more effectively than gauze by promoting re-epithelialization and collagen deposition as well as by stimulating neovascularization. The results of the wound healing process showed that DA of the chitin nonwoven fabrics had a profound effect on promoting wound healing. Notably, the regenerated chitin nonwoven fabrics with 71% DA significantly improved the wound healing compared to the commercial wound dressing Algoplaque film. Therefore, the regenerated chitin nonwoven fabrics are promising candidates for wound healing.

Review: Advances in preparation of chitooligosaccharides with heterogeneous sequences and their bioactivity.

Chitooligosaccharides has attracted increasing attention due to their diverse bioactivities and potential application. Previous studies on the bioactivity of chitooligosaccharides were mostly carried out using a mixture. The structure-function relationship of chitooligosaccharides is not clear. Recently, it is confirmed that chitooligosaccharides with different degrees of polymerization play different roles in many bioactivities. However, heterogeneous chitooligosaccharides with a single degree of polymerization is still a mixture of many uncertain sequences and it is difficult to determine which structure is responsible for biological effects. Therefore, an interesting and challenging field of studying chitooligosaccharides with heterogeneous sequences has emerged. Herein, we reviewed the current methods for preparing heterogeneous chitooligosaccharides, including chemical synthesis, separation techniques and enzymatic methods. Advances in the bioactivities of chitooligosaccharides with heterogeneous sequences are also reviewed.

Compromised chitin synthesis in lager yeast affects its Congo red resistance and release of mannoproteins from the cells.

A mutant lager strain resistant to the cell wall-perturbing agent Congo red (CR) was isolated and the genetic alterations underlying CR resistance were investigated by whole genome sequencing. The parental lager strain was found to contain three distinct Saccharomyces cerevisiae (Sc)-type CHS6 (CHitin Synthase-related 6) alleles, two of which have one or two nonsense mutations in the open reading frame, leaving only one functional allele, whereas the functional allele was missing in the isolated CR-resistant strain. On the other hand, the Saccharomyces eubayanus-type CHS6 alleles shared by both the parental and mutant strains appeared to contribute poorly to chitin synthase-activating function. Therefore, the CR resistance of the mutant strain was attributable to the overall compromised activity of CHS6 gene products. The CR-resistant mutant cells exhibited less chitin production on the cell surface and smaller amounts of mannoprotein release into the medium. All these traits, in addition to the CR resistance, were complemented by the functional ScCHS6 gene. It is of great interest whether the frequent nonsense mutations found in ScCHS6 open reading frame in lager yeast strains are a consequence of the domestication process of lager yeast.

Chemoenzymatic Synthesis of Chito-oligosaccharides with Alternating N-d-Acetylglucosamine and d-Glucosamine.

Chito-oligosaccharides (CHOS) are homo- or hetero-oligomers of N-acetylglucosamine (GlcNAc, A) and d-glucosamine (GlcN, D). Production of well-defined CHOS-mixtures, or even pure CHOS, with specific lengths and sugar compositions, is of great interest since these oligosaccharides have interesting bioactivities. While direct chemical synthesis of CHOS is not straightforward, chemo-enzymatic approaches have shown some promise. We have used engineered glycoside hydrolases to catalyze oligomerization of activated DA building blocks through transglycosylation reactions. The building blocks were generated from readily available (GlcNAc)2-para-nitrophenol through deacetylation of the nonreducing end sugar with a recombinantly expressed deacetylase from Aspergillus niger (AnCDA9). This approach, using a previously described hyper-transglycosylating variant of ChiA from Serratia marcescens (SmChiA) and a newly generated transglycosylating variant of Chitinase D from Serratia proteamaculans (SpChiD), led to production of CHOS containing up to ten alternating D and A units [(DA)2, (DA)3, (DA)4, and (DA)5]. The most abundant compounds were purified and characterized. Finally, we demonstrate that (DA)3 generated in this study may serve as a specific inhibitor of the human chitotriosidase. Inhibition of this enzyme has been suggested as a therapeutic strategy against systemic sclerosis.

A review on the synthesis of graft copolymers of chitosan and their potential applications.

Chitosan is an antimicrobial, biodegradable and biocompatible natural polymer, commercially derived from the partial deacetylation of chitin. Currently modified chitosan has occupied a major part of scientific research. Modified chitosan has excellent biotic characteristics like biodegradation, antibacterial, immunological, metal-binding and metal adsorption capacity and wound-healing ability. Chitosan is an excellent candidate for drug delivery, food packaging and wastewater treatment and is also used as a supporting object for cell culture, gene delivery and tissue engineering. Modification of pure chitosan via grafting improves the native properties of chitosan. Chitosan grafted copolymers exhibit high significance and are extensively used in numerous fields. In this review, modifications of chitosan through several graft copolymerization techniques such as free radical, radiation, and enzymatic were reported and the properties of grafted chitosan were discussed. This review also discussed the applications of grafted chitosan in the fields of drug delivery, food packaging, antimicrobial, and metal adsorption as well as dye removal.

Enzymatic preparation of chitooligosaccharides and their anti-obesity application.

Chitooligosaccharides (COS) are derived from chitosan, which can be used as nutraceuticals and functional foods. Because of their various biological activities, COS are widely used in the food, medicine, agriculture, and other fields. COS were prepared by chitosanase  from Pseudoalteromonas sp. SY39 and their anti-obesity activity was researched in mice in this study. The effects of hydrolysis time, temperature, the ratio of enzyme to chitosan, and pH on the productivity of COS were discussed. Preparation process of COS was established in a 5-L fermenter. COS were characterized and their anti-obesity activity was studied in animal experiments. The results showed that COS could effectively reduce serum lipid levels and obesity in mice, and have a good anti-obesity activity. The preparation technology and remarkable anti-obesity activity of COS further expand their applications in the food and pharmaceutical industries.

Robust enzymatic-mass spectrometric fingerprinting analysis of the fraction of acetylation of chitosans.

We developed a rapid and precise method to determine the fraction of acetylation (FA) of unknown chitosan samples using a combination of enzymatic sample hydrolysis, isotopic labeling, and HILIC-ESI-MS analysis. Chitosans are ß-(1,4)-linked, partially N-acetylated and linear polyglucosamines representing an interesting group of functional biopolymers with a broad range of applications. For a better understanding of their structure-function relationships, it is key to have sensitive, accurate structural analysis tools available to determine parameters like the degree of polymerization (DP), fraction of acetylation (FA), or pattern of acetylation (PA). Here, we describe an improved enzymatic/mass spectrometric method for FA analysis of chitosan polymers. In contrast to the original chitinosanase-based mass spectrometric fingerprinting analysis of FA, the new method is independent of the PA and the intermolecular variation in FA (DFA) of the chitosan sample. This allows accurate analysis of heterogeneously de-N-acetylated samples representing the majority of commercially available chitosans.

Polyphosphonium-oligochitosans decorated with nanosilver as new prospective inhibitors for common human enteric viruses.

The main objective of this work to explore new safe antiviral agents against hepatitis A virus (HAV), norovirus (NoV) and Coxsackievirus B4 (CoxB4) infections. In this context, we have successfully prepared new polyquaternary phosphonium oligochitosans (PQPOC1,2) to use them as natural synergistic in-situ bioreductants of silver ions into nanosilver and stabilizing agent for these nanosilver to fabricate PQPOCs-AgNPs nano-biocomposites (NBC1,2). The antiviral performance of the PQPOCs and NBCs against FCV, HAV, and CoxB4 reflects great virucidal activities for NBCs as compared with PQPOCs with maximum viral reduction% (41.42, 80.62, and 84.04%) for NBC1 against FCV, HAV, and CoxB4, respectively. Furthermore, the antiviral activity of NBC1 is concentration- / pH-dependent where NBC1 acquired its maximum antiviral at [NBC1] = 200 µL/mL and pH 4. Based upon these facts, we could attribute the enhanced virucidal efficacy of NBC1: (i) binding of AgNPs to the virions active sites. (ii) Electrostatic interaction between the positive brushes of PQPOC and negative targets of viruses. (iii) Inducing ribonuclease catalyzed by CS to degrade the viral RNA and consequently prevents its transcription and translation.

Synthesis of magnetic chitin-adsorbent for specific proteins.

A simple, convenient and inexpensive method for the preparation of magnetic chitin composite, in which magnetite particles are densely covered with the polysaccharide shell has been developed. Two-step procedure for magnetic chitin preparation includes: (i) adsorption of chitosan onto magnetite particles and (ii) N-selective acetylation of chitosan to produce magnetic chitin. The composite combines the magnetic properties of magnetite and the adsorption properties of chitin. The synthesized magnetic chitin is an efficient adsorbent of ß-d-GlcNAc-specific lectins and lysozyme. The adsorption capacity of magnetic chitin for wheat germ (Triticum vulgaris) and potato (Solanum tuberosum) lectins was in the range of 67-86 mg g-1 of the adsorbent. Magnetic chitin showed the high capacity for enzyme-lysozyme. Synthesized adsorbent is environmentally friendly and recyclable. Magnetic chitin may be used as a promising multi-purpose adsorbent of pollutants of organic or inorganic nature.

Preparation, characterization and application of rod-like chitin nanocrystal by using p-toluenesulfonic acid/choline chloride deep eutectic solvent as a hydrolytic media.

Chitin nanocrystal (ChiNC) was fabricated based on p-toluenesulfonic acid -choline chloride deep eutectic solvent treatment. The obtained ChiNC was about 12-44 nm in width and 206-399 nm in length. The crystalline structure and the functional groups of ChiNC were maintained during the preparation process. Moreover, porcine pancreas lipase (PPL) was successfully immobilized onto the ChiNC to form the immobilized PPL (PPL@ChiNC). The resulting PPL@ChiNC has enzyme loading and activity recovery of 35.6 mg/g and 82.5%, respectively. The thermal stability, pH and temperature adaptabilities of PPL@ChiNC was improved, comparing with free PPL. The demonstrated DES treatment process was efficient for ChiNC preparation and the as-prepared ChiNC exhibited great potentials in biocatalysis and biomedical field.

Robust chitin films with good biocompatibility and breathable properties.

Chitin is the second abundant polysaccharide after cellulose, and has been demonstrated to possess various applications in the fields of the biomaterials, water treatment, etc. Here, mechanically strong chitin films were successfully fabricated from chitin solution in NaOH/urea aqueous system with cooling by chemical crosslinking with epichlorohydrin (ECH). The results indicated that tensile strength and elongation at break of the chitin film prepared by using the weight ratio of ECH to chitin solution of 6:100 enhanced significantly to achieve 139 MPa and 43%, respectively, showing highly strong strength and elongation. The cell assay results confirmed that the chitin films were non-toxicity. Water vapor transmittance rate testing indicated that the chitin films had better breathable properties than commercial product. The robust chitin films with high transmittance, biocompatibility and breathability have great potentials in the wide applications such as biomedical, food packaging, wound healing, garments fields, etc.

Design and Synthesis of a Chitodisaccharide-Based Affinity Resin for Chitosanases Purification.

Chitooligosaccharides (CHOS) have gained increasing attention because of their important biological activities. Enhancing the efficiency of CHOS production essentially requires screening of novel chitosanase with unique characteristics. Therefore, a rapid and efficient one-step affinity purification procedure plays important roles in screening native chitosanases. In this study, we report the design and synthesis of affinity resin for efficient purification of native chitosanases without any tags, using chitodisaccharides (CHDS) as an affinity ligand, to couple with Sepharose 6B via a spacer, cyanuric chloride. Based on the CHDS-modified affinity resin, a one-step affinity purification method was developed and optimized, and then applied to purify three typical glycoside hydrolase (GH) families: 46, 75, and 80 chitosanase. The three purified chitosanases were homogeneous with purities of greater than 95% and bioactivity recovery of more than 40%. Moreover, we also developed a rapid and efficient affinity purification procedure, in which tag-free chitosanase could be directly purified from supernatant of bacterial culture. The purified chitosanases samples using such a procedure had apparent homogeneity, with more than 90% purity and 10⁻50% yield. The novel purification methods established in this work can be applied to purify native chitosanases in various scales, such as laboratory and industrial scales.

The study of inhibitory effects and mechanism of carboxylate chitooligomer on melanin, prepared by laccase/TEMPO system.

A carboxylate chitooligomer (C-COS) containing carboxyl groups attached to chitooligomer (COS) molecules has been prepared by laccase/2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) system, which is a green-chemistry method. Several experiments were designed to evaluate inhibition effects on melanin and mechanisms of C-COS. The results indicated that C-COS exhibited more distinct anti-melanogenic effects compared to COS. C-COS inhibits melanin production with tyrosine (Tyr) and DOPA as the substrate of melanin formation, and the inhibition rates are, respectively, 89.07% and 84.45%, which reach 1.4-2 times those of COS. UV-vis spectroscopy was used to elucidate the interaction mechanism between C-COS and tyrosinase (TYR). It is C-COS chelating with metal Cu ions in tyrosinase (TYR) that decreases the enzyme activity. Half-maximal inhibitory concentrations (IC50) of C-COS were calculated as 13.49 and 4.07 mg/mL for monophenolase (cresolase) and diphenolase (catecholase), respectively.

A novel thermophilic exochitinase ChiEn3 from Coprinopsis cinerea exhibits a hyperhydrolytic activity toward 85% deacetylated chitosan and a significant application to preparation of chitooligosaccharides from the chitosan.

ChiEn3 from Coprinopsis cinerea was characterized as an exo-acting chitinase with a processivity. ChiEn3 hydrolyzed only soluble chitin and exhibited a hyperhydrolytic activity toward 85% deacetylated chitosan which was 33.6-fold higher than its hydrolytic activity toward glycol chitin. Its maximum hydrolytic activity was observed at 60 °C and retained 66.2% of hydrolytic activity after 60 min incubation at 60 °C. Commercial 85% deacetylated chitosan was degraded by ChiEn3 to a series of COSs with a DP of 2-20 in which COSs with a DP of 3-6 were dominant, whereas, lab-prepared chitosan (FA = 0.65) was degraded by ChiEn3 to COSs with a DP of 2-10 in which the AA dimer was dominant. DPPH-radical-scavenging activity of ChiEn3-digested products of 85% deacetylated chitosan was 3.32-fold higher than that of undigested 85% deacetylated chitosan. Therefore, ChiEn3 shows a valuable advantage for application to the preparation of COSs from commercial 85% deacetylated chitosan.

Linolenic acid-modified methoxy poly (ethylene glycol)-oligochitosan conjugate micelles for encapsulation of amphotericin B.

Introduction of linolenic acid (LNA) and methoxy poly (ethylene glycol) (MPEG) to the backbone of oligochitosan (CS) afforded LNA-modified MPEG-CS conjugate (MPEG-CS-LNA). Amphotericin B-loaded MPEG-CS-LNA micelles (AmB-M) were prepared via dialysis method with 82.27 ± 1.96% of drug encapsulation efficiency and 10.52 ± 0.22% of drug loading capacity. The AmB-M enhanced AmB's water-solubility to 1.64 mg/mL, being 1640-folds higher than native AmB. The AmB-M obviously reduced hemolytic effect and renal toxicity of AmB when compared to marketed AmB injection (AmB-I). Its antifungal activity against Candida albicans was equivalent to AmB-I although AmB's release from AmB-M was significantly retarded. According to fluorescence microscopy test, the unchanged activity should be attributed to enhanced fungal cellular uptake of AmB-M caused by combined inducement of LNA and CS. The pharmacokinetic studies demonstrated that AmB-M also improved the pharmacokinetic parameters of AmB with AmB-I as control. Conclusively, developed LNA-modified MPEG-CS micellar system could be a viable alternative to the current toxic commercial AmB-I as a highly efficacious drug delivery system.