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The chemistry of chitosan is a promising way to afford biobased and biodegradable complex materials using highly reactive compounds such as anhydrides. However, the potential applications are limited due to the absence of control over the acylation and the lack of precise characterization. After a model study on glucosamine, a selective acylation method of amines or alcohols of chitosan oligomers using anhydrides has been developed. The N-acylation of chitooligosaccharides has been achieved using several anhydrides and the O-acylation has been achieved in three steps: (1) Amine protection using p-anisaldehyde (2) O-acylation (with four anhydrides), and (3) Amine deprotection by removing the protective anisyl groups. Functionalized chitooligosaccharides have been characterized by a precise quantification method using 13C nuclear magnetic resonance spectroscopy. The resulting degrees of substitution show in most of the cases a stochiometric reaction between anhydrides and amines of chitooligosaccharides, and around 70 % of efficiency for O-acylation, proving the promising potential of such modifications. To the best of our knowledge, we describe here the first example of selective acylation of chitooligosaccharides using anhydrides, and most of all the first example of 13C quantitative NMR spectroscopy performed on chitooligosaccharides and its derivatives. These innovative structures are the gateway to the creation of new biosourced and/or biodegradable surfactants.

Abstract Purpose The present research work focuses on the extraction of chitosanase enzyme from soil fungi. Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides which are new biomaterials that have many biological activities such as antitumour, antioxidant, antidiabetic and antimicrobial. Method A strain producing chitosanase was screened and identified as Fusarium oxysporum D18 with an accession number OL343607. Various physiological parameters (incubation type, carbon source, additive nitrogen source, statistical evaluation, solid state fermentation) were assessed to increase chitosanase production. Results Fusarium oxysporum D18 produced a considerable value of chitosanase (1.220 U/ml). After 7 days of incubation, the best carbon source was lactose, and the best nitrogen source was ammonium chloride. Statistical evaluation was carried out by using Plackett–Burman and Box-Behnken designs. The highest chitosanase production (1.994 U/ml) was induced by the medium composition g/l: KH2PO4 (1.5), MgSO4 (0.269), lactose (18), NH4Cl (1.26), pH (6.68), using a 5-day-old inoculum and chitosanase activity was 1.63 folds that of the original medium. The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied and the best solid substrate for higher chitosanase activity (2.246 U/ml) was raw shrimp heads and shells and chitosanase activity was 1.13 folds that of the optimized liquid cultures. An extracellular chitosanase was isolated and partially purified by using 75% saturation of ammonium sulphate. The highest chitosanase activity (3.667 U/ml) with a specific activity of 0.390 U/mg protein was obtained at enzyme protein concentration of 9.391 mg/ml, substrate concentration of 1.2 % (w/v), V max of the enzyme of approximately 0.430 U/mg protein, and K M of 0.26 % (w/v), at pH 5.6 and reaction temperature of 50 °C. The activity of the purified and characterized chitosanase increased by 3 times than that the original isolate activity. The enzyme was thermostable and retained about 55% of its original activity after heating at 70 °C for 15 min. The enzyme preparations were activated by Ca2+ ions and inactivated by Zn+2, Cu+2 ions, and EDTA. Conclusion An antitumour activity of chitooligosaccharides produced by the chitosanase was applied to the MCF-7 (breast carcinoma cells) and they had a cytotoxicity inhibitory effect against them about IC50 = 448 μg/ml.

Purpose: The present research work focuses on the extraction of chitosanase enzyme from soil fungi. Chitosan hydrolysis by chitosanase is one of the most effective methods to produce chitosan oligosaccharides which are new biomaterials that have many biological activities such as antitumour, antioxidant, antidiabetic and antimicrobial. Method: A strain producing chitosanase was screened and identified as Fusarium oxysporum D18 with an accession number OL343607. Various physiological parameters (incubation type, carbon source, additive nitrogen source, statistical evaluation, solid state fermentation) were assessed to increase chitosanase production. Results: Fusarium oxysporum D18 produced a considerable value of chitosanase (1.220 U/ml). After 7 days of incubation, the best carbon source was lactose, and the best nitrogen source was ammonium chloride. Statistical evaluation was carried out by using Plackett–Burman and Box-Behnken designs. The highest chitosanase production (1.994 U/ml) was induced by the medium composition g/l: KH2PO4 (1.5), MgSO4 (0.269), lactose (18), NH4Cl (1.26), pH (6.68), using a 5-day-old inoculum and chitosanase activity was 1.63 folds that of the original medium. The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied and the best solid substrate for higher chitosanase activity (2.246 U/ml) was raw shrimp heads and shells and chitosanase activity was 1.13 folds that of the optimized liquid cultures. An extracellular chitosanase was isolated and partially purified by using 75% saturation of ammonium sulphate. The highest chitosanase activity (3.667 U/ml) with a specific activity of 0.390 U/mg protein was obtained at enzyme protein concentration of 9.391 mg/ml, substrate concentration of 1.2 % (w/v), Vmax of the enzyme of approximately 0.430 U/mg protein, and KM of 0.26 % (w/v), at pH 5.6 and reaction temperature of 50 °C. The activity of the purified and characterized chitosanase increased by 3 times than that the original isolate activity. The enzyme was thermostable and retained about 55% of its original activity after heating at 70 °C for 15 min. The enzyme preparations were activated by Ca2+ ions and inactivated by Zn+2, Cu+2 ions, and EDTA. Conclusion: An antitumour activity of chitooligosaccharides produced by the chitosanase was applied to the MCF-7 (breast carcinoma cells) and they had a cytotoxicity inhibitory effect against them about IC50 = 448 μg/ml. Highlights: (1) We screened and identified a chitosanase producing strain from the soil as Fusarium oxysporum D18 with an accession number OL343607. (2) The enzyme activity of chitosanase was greatly improved by optimizing the fermentation medium of the fungus. (3) The activity of chitosanase was optimized by statistical evaluation that was carried out by using Plackett-Burman and Box-Behnken designs. (4) The production of chitosanase by Fusarium oxysporum D18 in solid state cultures using different solid substrates was studied for increasing activity. (5) Partial purification of the enzyme was performed by using 75 % saturation of ammonium sulphate. (6) Physical and chemical properties of the enzyme were performed leading to a highly thermostable chitosanase. (7) An antitumour chitosanase activity was applied to the MCF-7. [ABSTRACT FROM AUTHOR]

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomertriggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity. [ABSTRACT FROM AUTHOR]

The establishment of defense reactions to protect plants against pathogens requires the recognition of invasion patterns (IPs), mainly detected by plasma membrane-bound pattern recognition receptors (PRRs). Some IPs, also termed elicitors, are used in several biocontrol products that are gradually being developed to reduce the use of chemicals in agriculture. Chitin, the major component of fungal cell walls, as well as its deacetylated derivative, chitosan, are two elicitors known to activate plant defense responses. However, recognition of chitooligosaccharides (COS) in Vitis vinifera is still poorly understood, hampering the improvement and generalization of protection tools for this important crop. In contrast, COS perception in the model plant Arabidopsis thaliana is well described and mainly relies on a tripartite complex formed by the cell surface lysin motif receptor-like kinases (LysM-RLKs) AtLYK1/CERK1, AtLYK4 and AtLYK5, the latter having the strongest affinity for COS. In grapevine, COS perception has for the moment only been demonstrated to rely on two PRRs VvLYK1-1 and VvLYK1-2. Here, we investigated additional players by overexpressing in Arabidopsis the two putative AtLYK5 orthologs from grapevine, VvLYK5-1 and VvLYK5-2. Expression of VvLYK5-1 in the atlyk4/5 double mutant background restored COS sensitivity, such as chitin-induced MAPK activation, defense gene expression, callose deposition and conferred non-host resistance to grapevine downy mildew (Erysiphe necator). Protein-protein interaction studies conducted in planta revealed a chitin oligomer-triggered interaction between VvLYK5-1 and VvLYK1-1. Interestingly, our results also indicate that VvLYK5-1 mediates the perception of chitin but not chitosan oligomers showing a part of its specificity.

Burns can impair the barrier function of the skin, and small burns can also cause high mortality. The WHO has described that over 180,000 people die of burns worldwide each year. Thus, the treatment of burn wounds is a major clinical challenge. Chitooligosaccharides (COS) are alkaline amino oligosaccharides with small molecular weights obtained by enzyme or chemical degradation of chitosan. With the characteristics of biocompatibility, water solubility and degradability, it has attracted increasing attention in the fields of biomedicine. In the present study, we used COS to treat deep second-degree burn wounds of rat skin and found that COS was able to promote wound healing. We also revealed that COS could promote fibroblast proliferation. Transcriptome sequencing analysis was performed on COS-treated fibroblasts to identify the underlying mechanisms. The results showed that COS was able to promote wound healing through regulation of the mitogen-activated protein kinase (MAPK) pathway and growth factor Hepatocyte Growth Factor (HGF). Our results provide a potential drug for burn wound therapy and the related molecular mechanism.

The present study investigated the interactions between chitooligosaccharides (COS) and metal ions (Fe, Zn, and Mg) through the analysis of molecular dynamics (MD) simulations. The results indicate that each of the Fe, Zn, and Mg ions formed complexes with 1.35, 1.32, and 1.37 COS molecules, whereas each of the COS combined with 2.22 of Fe, 2.21 of Zn, and 2.37 of Mg ions, respectively. Two coordination patterns with COS to Fe/Zn/Mg ratios of 1:1 or 2:1 were observed in the complex systems and formed a bridge and/or pendant structure. The nitrogen atoms on the amino groups and the oxygen atoms on the hydroxyl groups of the COS chain combined with the Fe/Zn/Mg ions through the formation of metal-N and metal-O coordination bonds. The stability of the generated COS-Fe/Zn/Mg complexes were characterized in the following order based on the calculations of their respective molecule interaction energies: COS-Zn > COS-Fe > COS-Mg. [ABSTRACT FROM AUTHOR]

Chitooligosaccharides (COS) have many bioactive functions and favorable prospects in the fields of biomedicine and functional foods. In this study, COS was found to significantly improve the survival rate of neonatal necrotizing enterocolitis (NEC) model rats, alter the composition of the intestinal microbiota, inhibit the expression of inflammatory cytokines, and alleviate intestinal pathological injury. In addition, COS also increased the abundance of Akkermansia , Bacteroides , and Clostridium sensu stricto 1 in the intestines of normal rats (the normal rat model is more universal). The in vitro fermentation results found that COS was degraded by the human gut microbiota to promote the abundance of Clostridium sensu stricto 1 and produced numerous short-chain fatty acids (SCFAs). In vitro metabolomic analysis revealed that COS catabolism was associated with significant increases in 3-hydroxybutyrate acid and γ-aminobutyric acid. This study provides evidence for the potential of COS as a prebiotic in food products and to ameliorate NEC development in neonatal rats. [Display omitted] [ABSTRACT FROM AUTHOR]

Antifungal activity against the dermatophytic fungus Trichophyton rubrum by a well-characterized chitooligosaccharides (COS) sample, hydrolyzed using a recombinant chitosanase, was investigated in vitro and in vivo . The minimum inhibitory concentration (MIC) of COS ranged between 0.25 and 0.50%, which was measured using a microdilution method. Analysis of inhibition rates using an agar diffusion method showed that treatment with 0.5% and 1% COS significantly suppressed T. rubrum cell growth ( p < 0.05 and p < 0.01, respectively, in comparison with untreated control). Morphological changes and structural alterations of cells were observed by TEM. In vivo efficacy of COS in treatment of T. rubrum dermatophytosis was evaluated using a guinea pig model. Skin lesion scores revealed a strong, dose-dependent therapeutic effect of COS. The 5% COS group showed a reduction of skin lesions even greater than that of the positive control group treated with 1% fluconazole (FCZ). Histological analysis revealed no inflammation or tissue destruction in the groups treated with 5% COS or 1% FCZ. Hyperkeratosis was also observed, perhaps resulting from a defensive response of the tissue cells to COS. The findings indicate that COS has excellent potential for development of novel antifungal drugs for clinical treatment/remission of dermatophytoses. [ABSTRACT FROM AUTHOR]

Burns can impair the barrier function of the skin, and small burns can also cause high mortality. The WHO has described that over 180,000 people die of burns worldwide each year. Thus, the treatment of burn wounds is a major clinical challenge. Chitooligosaccharides (COS) are alkaline amino oligosaccharides with small molecular weights obtained by enzyme or chemical degradation of chitosan. With the characteristics of biocompatibility, water solubility and degradability, it has attracted increasing attention in the fields of biomedicine. In the present study, we used COS to treat deep second-degree burn wounds of rat skin and found that COS was able to promote wound healing. We also revealed that COS could promote fibroblast proliferation. Transcriptome sequencing analysis was performed on COS-treated fibroblasts to identify the underlying mechanisms. The results showed that COS was able to promote wound healing through regulation of the mitogen-activated protein kinase (MAPK) pathway and growth factor Hepatocyte Growth Factor (HGF). Our results provide a potential drug for burn wound therapy and the related molecular mechanism., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Zhang, Zhang, Yang, Qiao, Wang, Yan, Li, Huang, Zhou, Hu and Zhang.)

Abstract: In the present research, chitooligosaccharides (COS) with molecular weight 800–3000Da and 90% of deacetylation were chemically modified by grafting 2-chloroethylamino hydrochloride at C-6 position to synthesize angiotensin I converting enzyme (ACE) inhibitory chitin derivatives based on the properties of ACE inhibitors. The synthetic product was designated as aminoethyl chitooligosaccharide (AE-COS) with molecular weight 800.79–4765Da. Its IC50 value on ACE was 0.8017μg/mL. Furthermore, Lineweaver-Burk plots revealed that the inhibition was competitive via obligatory binding site of ACE. Therefore, these results exhibited that substitution of the hydrogen atom at the C-6 position of pyranose residue by the aminoethyl group promotes ACE inhibitory effects of COS. [Copyright &y& Elsevier]

Abstract: Despite extensive study on biological activities of chitosan and chitooligosaccharides (COS), there is no experimental evidence available as to COS mediated inhibition of free radical damage in cellular oxidizing systems. In this study, radical scavenging efficacies of different molecular weight bearing COS were assessed and their intracellular radical scavenging effects were tested employing B16F1, murine melanoma cell line. The results exhibited appreciable suppression in occurrence of intracellular radical species in the presence of low molecular weight bearing COS (<1 kDa) confirming low molecular weight is important for observed activities in biological systems. However, DNA oxidation carried out in the presence of COS clearly exhibited that COS exert protective effect on oxidative damage of purified genomic DNA regardless of molecular weight. Low molecular weight bearing COS was observed to be successively participated in suppression of NF-κB gene promoter activity suggesting its capability to prevent oxidative stress related disease complications. Moreover, induction of intracellular glutathione (GSH) level in the presence of COS promoted the effectiveness of COS to act against cellular oxidative stress. [Copyright &y& Elsevier]

Abstract: Even though several studies report the importance of chitosan derivatives for their anticancer activity, no clear information is available to describe the relationship between their charge properties and observed activities. In this research, differently charged chitooligosaccharide (COS) derivatives were synthesized and their anticancer activities were studied using three cancer cell lines, HeLa, Hep3B and SW480. Neutral red and MTT cell viability studies revealed that, highly charged COS derivatives could significantly reduce cancer cell viability, regardless to the positive or negative charge. Further, fluorescence microscopic observations and DNA fragmentation studies confirmed that the anticancer effect of these highly charged COS derivatives were due to necrosis. However, the exact molecular mechanism for anticancer activity of strongly charged COS compared to their poorly charged counterparts is not clear. [Copyright &y& Elsevier]

Abstract: Two chitooligosaccharides (COS) derivatives were synthesised by introducing carboxyl (–COCH2CH2COO−) and quaternized amino groups to the amino position of COS with different substitution degrees (CCOS-1–3 and QCOS-1–3) for the purpose of altering total amount of hydrogen atoms capable of reacting with radicals and modifying their metal ion chelating ability. Scavenging of carbon-centered and DPPH radicals was directly affected by the amount of abstractable hydrogen atoms in COS molecules. In contrast, structure-activity relationships revealed that chelation of Fe2+ ions indirectly contributed for their observed hydroxyl radical scavenging activity apart from hydrogen abstraction. [Copyright &y& Elsevier]

Abstract: In the present research, chitooligosaccharides (COS) were carboxylated with –COCH2CH2COO− groups to obtain specific structural features similar to Captopril®. Angiotensin I converting enzyme (ACE) inhibitory activity of carboxylated COS was studied and observed to enhance its activity with increased substitution degree. Further, Lineweaver–Burk plot analysis revealed that inhibition was competitive via obligatory binding site of the enzyme. This was accompanied with substitution of positively charged quarternized amino groups to COS with different substitution degrees, in which negative impact on ACE inhibition was observed. [Copyright &y& Elsevier]