Chemical Deacetylation Research Articles

Chitin and Chitosan Production from Shrimp Wastes by a Two-Step Process Consisting of Molasses-Based Lactic Fermentation and Chemical Deacetylation: Insights into the Antimicrobial, Microsphere and Film-Forming Properties of Chitosan

Chitin and Chitosan Production from Shrimp Wastes by a Two-Step Process Consisting of Molasses-Based Lactic Fermentation and Chemical Deacetylation: Insights into the Antimicrobial, Microsphere and Film-Forming Properties of Chitosan

Enzymatic hydrolysis of chitinous wastes pretreated by deep eutectic solvents into N-acetyl glucosamine

In this study, we present an efficient and green extraction-pretreatment integrated approach for enhancing enzymatic conversion of chitinous wastes into N-acetyl-d-glucosamine (GlcNAc). Firstly, the enzyme cocktail containing a chitinase CmChi1 and a N-acetyl glucosaminase CmNAGase were constructed for hydrolyzing chitin into sole GlcNAc. Secondly, deep eutectic solvent (DES), consisting of choline chloride and glycollic acid was used to treat chitinous wastes. Under optimal conditions, chitin yield reach to 72 % with a purity of 98 %. Fourier-transform infrared spectroscopy, thermogravimetric analysis, and X-ray diffraction analysis revealed that the crystallinity and thermal stability of the obtained chitin decreased upon DES treatment without alteration of the chemical structure or deacetylation. Finally, the concentration of GlcNAc was increased 2–6 folds by enzymatic hydrolysis of DES-treated chitinous wastes (including shrimp shell, crab shell, ganoderma spores wall, and mycelium). The process provides a promising strategy for degrading chitinous wastes to produce high valued GlcNAc.

Synthesis and characterization of poly (acrylamide‐co‐acrylic acid)/chitosan (derived from fish scales) for controlled release of amoxicillin drug

AbstractChitosan (CS) can be extracted from waste fish scales through a series of chemical processes, including demineralization, deproteinization, and deacetylation. The biomaterial poly (acrylamide [AM]‐co‐acrylic acid [AA])/CS is a unique superabsorbent material synthesized through the free radical graft copolymerization method by using AM, AA, and CS powder. During synthesis, ammonium persulfate is used as an initiator, and N, N′‐methylene bisacrylamide is used as a cross‐linker. Scanning electron microscopy and Fourier transform infrared spectroscopy have been studied to know about the morphology and structure of the synthesized superabsorbent materials. The water absorbency of the material continuously increases from day 1 to day 30. The thermogravimetric analysis (TGA) and derivative thermogravimetry (DTG) of the superabsorbent material show adequate thermal stability. The X‐ray diffraction (XRD) analysis indicates that the synthesized biomaterial is virtually amorphous in nature. The percentage of biodegradability increases with time. The encapsulation efficiency of amoxicillin drugs is around 70%, which indicates that the biomaterial is highly efficient for drug release applications. The synthesized novel biomaterial is a low‐cost, eco‐friendly, and thermally stable hydrogel that can be effectively used in drug release applications.Highlights Chitosan (CS)‐based hydrogels are highly pH‐sensitive towards drug release. The biodegradability and swelling behavior of the synthesized biomaterial were studied by adopting a suitable methodology. The novel material formed was characterized by using Fourier transform infrared spectroscopy, TGA, and Scanning electron microscopy. The encapsulation efficiency, kinetic study, and controlled drug release behavior of the amoxicillin drug were studied in solutions of simulated gastric fluid and phosphate‐buffered saline. The material developed in this study shows good performance in controlled drug delivery, and is therefore suitable for manufacturing medical devices used in drug delivery.

Physicochemical Characterization and Antimicrobial Analysis of Vegetal Chitosan Extracted from Distinct Forest Fungi Species.

The main goal of this investigation is to conduct a thorough analysis of the physical, chemical, and morphological characteristics of chitosan derived from various forest fungi. Additionally, the study aims to determine the effectiveness of this vegetal chitosan as an antimicrobial agent. In this study, Auricularia auricula-judae, Hericium erinaceus, Pleurotus ostreatus, Tremella fuciformis, and Lentinula edodes were examined. The fungi samples were subjected to a series of rigorous chemical extraction procedures, including demineralization, deproteinization, discoloration, and deacetylation. Subsequently, the chitosan samples were subjected to a comprehensive physicochemical characterization analysis, encompassing Fourier transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDX), X-ray diffraction (XRD), degree of deacetylation determination, ash content determination, moisture content determination, and solubility determination. To evaluate the antimicrobial efficacy of the vegetal chitosan samples, two different sampling parameters were employed, namely human hand and banana, to assess their effectiveness in inhibiting microbial growth. Notably, the percentage of chitin and chitosan varied significantly among the distinct fungal species examined. Moreover, EDX spectroscopy confirmed the extraction of chitosan from H. erinaceus, L. edodes, P. ostreatus, and T. fuciformis. The FTIR spectra of all samples revealed a similar absorbance pattern, albeit with varying peak intensities. Furthermore, the XRD patterns for each sample were nearly identical, with the exception of the A. auricula-judae sample, which exhibited sharp peaks at ~37° and ~51°, while the crystallinity index of this same sample was approximately 17% lower than the others. The moisture content results indicated that the L. edodes sample was the least stable, while the P. ostreatus sample was the most stable, in terms of degradation rate. Similarly, the solubility of the samples showed substantial variation among each species, with the H. erinaceus sample displaying the highest solubility among the rest. Lastly, the antimicrobial activity of the chitosan solutions exhibited different efficacies in inhibiting microbial growth of skin microflora and microbes found on the peel of Musa acuminata × balbisiana.

Chitin and Chitosan Preparation from Malaysian Black Soldier Fly Biomass: A Preliminary Study

Chitin, as well as its deacetylated variant chitosan, has a wide range of applications in biomedical, agricultural, environmental and food industries. Currently, chitin is commercially produced from crustacean shells rather than fungal or insect sources. In this study, chitin from different lifecycle phases of the black soldier fly (BSF) was extracted and deacetylated into chitosan. Both BSF chitin and chitosan were further subjected to FTIR analysis. The chitin extraction yield % was obtained according to the following order: coccoon>larvae>prepupae. On the other hand, the yield of chemical deacetylation of chitin into chitosan showed a different trend: larvae>prepupae>coccoon. All samples have a deacetylation degree (DD) of more than 90 % except for chitosan from prepupae. FTIR profiles indicated all chitin extracted were in the α-form while the deacetylated chitosan matched closely to the commercially available chitosan. Overall, this study indicated that BSF biomass from different lifecycle phases could be a promising alternative resource for industrial chitin and chitosan production.

Chitosan Hydrogel for Removing of Heavy Metal Ions from Water: A Review

Industrial and agricultural discharges have been shown as important sources for a wide range of contaminants like heavy metals. This study focused on the assessment of the toxic effect of heavy metals and illustrates some methods of eliminating or avoiding these contaminants. In certain methods, heavy metals (Cu, Zn, Cr, Ni, Pb, Hg, and Cd) are recovered, and range of absorbents are tested. A number of studies have reported the complex interaction of polymers with metal ions with the polymers for eliminating heavy metals (e.g. cellulose, chitosan, and chitin. Metal ions are absorbed by these polymers by the amine and hydroxyl groups. They have unique features that incorporate polyxysalt arrangement, capacity to frame films, optical underlying qualities, and chelates metal particles. Chemical bonds, pH, crystallinity, and deacetylation are controlling the mechanism and performance of the absorption. Some methods increase the efficiency, selectivity, and quality of chitosan: the first is through chemical modification employing grafting and crosslinking, and the second by physical modification, such as manufacturing gel granules and reducing crystallization. This study aims to investigate the removal of heavy metals from water by the sorption method and to study and analyze literature dealing with this topic using chitosan hydrogel.

IL versus DES: Impact on chitin pretreatment to afford high quality and highly functionalizable chitosan

Chitin is mainly extracted from crustaceans, but this resource is seasonally dependent and can represent a major drawback to satisfy the traceability criterion for high valuable applications. Insect resources are valuable alternatives due to their lower mineral content. However, the deacetylation of chitin into chitosan is still an expensive process. Therefore, we herein compare the impact of both DES/IL-pretreatments on the efficiency of the chemical deacetylation of chitin carried out over two insect sources (Bombyx eri, BE and Hermetia illucens, HI) and shrimp shells (S). The results showed that chitosans obtained from IL-pretreated chitins from BE larva, present lower acetylation degrees (13–17%) than DES-pretreated samples (18–27%). A selective N-acylation reaction with oleic acid has also been performed on the purest and most deacetylated chitosans leading to high substitution degrees (up to 27%). The overall approach validates the proper chitin source and processing methodology to achieve high quality and highly functionalizable chitosan.

Biosynthesis and characterization of deuterated chitosan in filamentous fungus and yeast

Deuterated chitosan was produced from the filamentous fungus Rhizopus oryzae, cultivated with deuterated glucose in H2O medium, without the need for conventional chemical deacetylation. After extraction and purification, the chemical composition and structure were determined by Fourier-transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), and small-angle neutron scattering (SANS). 13C NMR experiments provided additional information about the position of the deuterons in the glucoseamine backbone. The NMR spectra indicated that the deuterium incorporation at the non-exchangeable hydrogen positions of the aminoglucopyranosyl ring in the C3 – C5 positions was at least 60–80 %. However, the C2 position was deuterated at a much lower level (6%). Also, SANS showed that the structure of deuterated chitosan was very similar compared to the non-deuterated counterpart. The most abundant radii of the protiated and deuterated chitosan fibers were 54 Å and 60 Å, respectively, but there is a broader distribution of fiber radii in the protiated chitosan sample. The highly deuterated, soluble fungal chitosan described here can be used as a model material for studying chitosan-enzyme complexes for future neutron scattering studies. Because the physical behavior of non-deuterated fungal chitosan mimicked that of shrimp shell chitosan, the methods presented here represent a new approach to producing a high quality deuterated non-animal-derived aminopolysaccharide for studying the structure-function association of biocomposite materials in drug delivery, tissue engineering and other bioactive chitosan-based composites.

Consequences of chemical deacetylation on physicochemical, structural and functional characteristics of chitosan extracted from deep-sea mud shrimp

Extraction of chitosan from deep-sea mud shrimp (Solenocera hextii) shell waste was conducted using a chemical method by altering the deacetylation times. The degree of deacetylation of extracted chitosan were 75.5 ± 0.36, 85 ± 0.38 and 87± 0.32 %, and the molecular weights were 263.95±1.16, 52.61± 0.44, and 5.15± 0.14 kDa respectively for CH-1.5, CH-3, and CH-6. Deacetylation time significantly influenced the structure and physicochemical properties of chitosan. SEM results exposed smooth granular surface structure and XRD peaks obtained were identical with commercial chitosan. Deacetylation time for CH-3 was found optimum for commercial extraction of this natural biopolymer resource. Water binding and fat binding capacities of the extracted deep-sea mud shrimp chitosan were significantly higher than commercial chitosan from coastal shrimp and are suitable for many functional food applications.

Chitin and Chitosan: Properties and Applications

Chitin/chitosan is the natural biocontrol active ingredient, found in the shells of crustaceans, exoskeleton of mollusks and cell walls of fungi. Chitin can be converted into chitosan by different chemical and enzymatic deacetylation. Chitin and chitosan are used as drug carriers owing to biocompatible, biodegradable and non-toxic, character. Chitosan is an antimicrobial agent with applications in agriculture, industry, food, biomedical and biotechnology fields. Chitosan also acts as elicitors and has an ability to form films. Chitosan also has some therapeutic activities like lowering cholesterol, anti-ulcer, wound healing and anti-tumor activity.

Production and physicochemical characterization of chitosan for the harvesting of wild microalgae consortia

The use of chitosan to harvest microalgae is a strategic step that seeks to reach an economically competitive price to recover lipids, proteins, and pigments. The aim of the present work was to design low-molecular-weight chitosan from shrimp shells and its physicochemical characterization, to be used for the harvesting of wild microalgae consortia. The chitosan was obtained by chemical deacetylation of shrimp shells, and physicochemical characterization was made using the instrumental methods DSC, TGA, X-ray, FTIR, and SEM. The harvesting of wild microalgae consortia was performed by the jar test method. The obtained chitosan had a low molecular weight (169 KDa), a deacetylation degree of 83 %, a decomposition temperature (TD) of 280 °C, and a crystallinity of 38.2 %. The microalgae genera found in the consortium were Scenedesmus sp., Chlorella sp., Schroderia sp., and Chlamydomonas sp. The microalgae removal efficiency of the chitosan was 99.2 % with 20 mg L−1.

Expression of Bacillus licheniformis chitin deacetylase in E. coli pLysS: Sustainable production, purification and characterisation.

Chitosan obtained by enzymatic deacetylation of chitin using chitin deacetylase (CDA) holds promise primarily due to the possibility to yield chitosan with non-random patterns of acetylation and more environmentally friendly process compared to chemical deacetylation. In the present study, a sustainable bioprocess is reported for over-expression of a bacterial CDA in E. coli pLysS cells. A Bacillus licheniformis CDA gene is identified in the genome of the bacterium, cloned, and expressed, yielding enzymatically active recombinant protein. For enzyme production, a growth medium is formulated using carbon and nitrogen sources, which do not compete with the human food chain. The maximum enzyme activity of 320 ± 20 U/mL is achieved under optimized conditions. The CDA productivity is improved by about 23 times in shake flask culture by optimizing operating conditions and medium components. The CDA is purified and the enzyme kinetic values i.e. Km, Vmax and Kcat are reported. Also the effect of cofactors, temperature, and pH on the enzyme activity is reported. Further, economic yield is proposed for production of CDA through this bioprocess.

An integral and sustainable valorisation strategy of squid pen by-products

The aim of this study was to present a sustainable valorisation approach of Loligo opalescens squid pen by-products by optimal production of chitin and chitosan biopolymers and recycling of the generated effluents. The integral recovery of squid pens started with the optimization of the deproteinization process to isolate chitin, including less hazardous enzymatic alternatives to conventional chemical treatments. Response surface methodology analysis yielded optimal conditions of 53.2 °C/pH 8.84 for Alcalase, 54.7 °C/pH 9.16 for Esperase and 0.2 M NaOH/27.5 °C for alkaline hydrolysis. The generated effluents were a good source of organic nitrogen (squid peptones) for the culture of a lactic acid bacterium (Pediococcus acidilactici), with biomass and metabolite productions comparable to commercial Man, Rogosa, Sharpe medium. Secondly, optimal process time and alkali concentration for the chemical deacetylation of chitin were established at 16 h/64% NaOH, obtaining high purity chitosan (deacetylation degrees >92%, molecular weights -Mn- ranging from 139 to 300 kDa). Optimized chitin and chitosan processes led to lower energetic costs. Alkaline effluents from chitosan production were applied as a reagent in the selective precipitation of chondroitin sulphate from enzymatic hydrolysates of shark cartilage by-products. Finally, the goodness of the proposed valorisation strategy was evaluated by developing a Material Flow Analysis.

Profiling Cytidine Acetylation with Specific Affinity and Reactivity.

The human acetyltransferase NAT10 has recently been shown to catalyze formation of N4-acetylcytidine (ac4C), a minor nucleobase known to alter RNA structure and function. In order to better understand the role of RNA acetyltransferases in biology and disease, here we report the development and application of chemical methods to study ac4C. First, we demonstrate that ac4C can be conjugated to carrier proteins using optimized protocols. Next, we describe methods to access ac4C-containing RNAs, enabling the screening of anti-ac4C antibodies. Finally, we validate the specificity of an optimized ac4C affinity reagent in the context of cellular RNA by demonstrating its ability to accurately report on chemical deacetylation of ac4C. Overall, these studies provide a powerful new tool for studying ac4C in biological contexts, as well as new insights into the stability and half-life of this highly conserved RNA modification. More broadly, they demonstrate how chemical reactivity may be exploited to aid the development and validation of nucleobase-targeting affinity reagents designed to target the emerging epitranscriptome.

Low Cost Chitosan Biopolymer for Environmental Use Made from Abundant Shrimp Wastes

Chitosan, an important biopolymer with many uses, is made by deacetylation of chitin. Classical hydrolysis at high temperatures (above 100 °C) in 50 % concentrated sodium hydroxide during several hours produces chitosan, a poly-beta-glucosamine. This chemical deacetylation step is often performed under pressure in boiling ethyleneglycol and tends to degrade polymer chain length, thus the interesting polymer properties. It is convenient to search for a more economical production pathway in order to make chitosan available for environmental applications, such as use in water suspended matter flocculation. Chitin was extracted from the exoskeletons of shrimps and its deacetylation kinetics was followed for 35 days at 20 and 35 °C. The results obtained using different techniques (conductivity, viscometry, 13C NMR and thermogravimetry) showed that the deacetylation degree of chitin increased with the incubation time reaching 99 and 88 % at 35 and 20 °C, respectively, after 35 days. Moreover, during the incubation time, the molecular weight of chitosan showed moderate decrease compared to deacetylated chitin at high temperature. Clarification or urban waste water with chitosan biopolymer demonstrated good results at 1.5 mg/L concentrations.

Identification of Alp1U and Lom6 as epoxy hydrolases and implications for kinamycin and lomaiviticin biosynthesis

The naturally occurring diazobenzofluorenes, kinamycins, fluostatins and lomaiviticins, possess highly oxygenated A-rings, via which the last forms a dimeric pharmacophore. However, neither the A-ring transformation nor the dimerization mechanisms have been explored thus far. Here we propose a unified biosynthetic logic for the three types of antibiotics and verify one key reaction via detailed genetic and enzymatic experiments. Alp1U and Lom6 from the kinamycin and lomaiviticin biosynthesis, respectively, are shown to catalyse epoxy hydrolysis on a substrate that is obtained by chemical deacetylation of a kinamycin-pathway-derived intermediate. Thus, our study provides the first evidence for the existence of an epoxy intermediate in lomaiviticin biosynthesis. Furthermore, our results suggest that the dimerization in the lomaiviticin biosynthesis proceeds after dehydration of a product generated by Lom6.

Acetylation of woody lignocellulose: significance and regulation

Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase) or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose toward improved saccharification. In this review we: (1) summarize literature on lignocellulose acetylation in different tree species, (2) present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, (3) describe plant proteins involved in lignocellulose O-acetylation, (4) give examples of microbial enzymes capable to de-acetylate lignocellulose, and (5) discuss prospects for exploiting these enzymes in planta to modify xylan acetylation.

Chemical deacetylation natural xanthan (Jungbunzlauer®)

With the aim of adapting a method for removal of acetyl groups from xanthan, reactions of chemical deacetylation were carried out with natural xanthan (Jungbunzlauer®) with variations on the following parameters: biopolymer and alkali concentration (sodium and potassium hydroxide). The deacetylation reaction was performed at 25 oC for three hours. The proposed methodology was efficient to promote the deacetylation reaction. The viscosity of xanthan increased when the alkali concentration was higher in the deacetylation reaction. Xanthan concentration in the solution used in the deacetylation reaction did not influence the solutions viscosity, as similar results in both tested biopolymer concentrations (0.5 and 1%) were obtained for all experiments in different shear rates. Deacetylation reactions at 25 oC for three hours with sodium and potassium hydroxide in 0.01 mol.L-1 showed a viscosity of 410 and 420 mPa.s at 10 s-1 and acetylation degree 1.3 e 1.4%, respectively.

General synthesis of mono-, di-, and tri-acetylated indoles from indolin-2-ones

Having developed the one-pot triacetylation of indolin-3-ones, we have now devised a simple two-step reaction sequences to produce di- and mono-acetylated indoles from indolin-2-ones. The indolin-2-ones were first subjected to acetylation in the presence of acetic anhydride and a catalytic amount of N, N-dimethylaminopyridine to give 2-acetoxy-1,3-diacetylindoles. Subsequently, an enzyme-assisted deacetylation resulted in the chemoselective deprotection of the acetoxy group to produce 1,3-diacetyl-2-hydroxyindoles. However, a chemical deacetylation of 2-acetoxy-1,3-diacetylinoles under mild basic or acidic conditions resulted in the formation of 3-acetyl-2-hydroxyindoles.

Genetic improvement of Bacillus licheniformis strains for efficient deproteinization of shrimp shells and production of high-molecular-mass chitin and chitosan.

By targeted deletion of the polyglutamate operon (pga) in Bacillus licheniformis F11, a derivative form, F11.1 (Δpga), was obtained that, along with lacking polyglutamate (PGA) formation, displayed enhanced proteolytic activities. The phenotypic properties were maintained in a strain in which the chiBA operon was additionally deleted: F11.4 (ΔchiBA Δpga). These genetically modified strains, carrying the Δpga deletion either alone (F11.1) or together with the ΔchiBA (F11.4) deletion, were used in fermentations (20-liter scale) aiming at the deproteinization of shrimp shells in order to obtain long-chain chitin. After chemical deacetylation, the resulting chitosan samples were analyzed by nuclear magnetic resonance spectroscopy, size exclusion chromatography, and viscometry and compared to a chitosan preparation that was produced in parallel by chemical methods by a commercial chitosan supplier (GSRmbH). Though faint lipid impurities were present in the fermented polysaccharides, the viscosity of the material produced with the double-deletion mutant F11.4 (Δpga ΔchiBA) was higher than that of the chemically produced and commercially available samples (Cognis GmbH). Thus, enhanced proteolytic activities and a lack of chitinase activity render the double mutant F11.4 a powerful tool for the production of long-chain chitosan.