Degradation of Chitin and Chitosan by a Recombinant Chitinase Derived from a Virulent <i>Aeromonas hydrophila</i>Isolated from Diseased Channel Catfish

A chitinase was identified in extracellular products of a virulent?Aeromonas hydrophila?isolated from diseased channel catfish (Ictalurus punctatus). Recombinant chitinase (rChi-Ah) was produced in?Escherichia coli. Purified rChi-Ah had optimal activity at temperature of 42℃?and pH 6.5. The affinity (Km) for chitosan was 4.18 mg·ml-1?with?Vmax?of 202.5 mg·min-1·mg-1. With colloidal chitin as substrate, rChi-Ah generated N,N’-diacetyl-glucosamine predominantly. Conversion of chitosan (≥75% deacetylated) by rChi-Ah revealed five major products: 2 to 4 units of glucosamine, all of which had at least one acetyl group. It was determined that N-acetylated glucosamine was the recognition and cleavage site of rChi-Ah;the minimal and maximal cleavages were two and four glucosamine units, respectively. Functional analysis of rChi-Ah suggests that?A. hydrophilachitinase is a bioactive chitinolytic enzyme, which may benefit the pathogen for survival and/or infection.

A Review of Various Sources of Chitin and Chitosan in Nature

Chitin was first discovered by its name from the Greek word“chiton”,which means“mail coat”.It is indeed a polysaccharide made up of naturally occurring acetyl-D-glucosamine monomers.Hatchett was the first researcher who extracted chitin from the shells of mollusks(crabs and lobsters),prawns,and crayfish in 1799.Later in 1811,Henri Braconnot discovered chitin in the cell walls of mushrooms and called it“fungine”.Chitin and chitosan are abundant in the biosphere as essential components of many organisms’exoskeletons and as by-products of the global seafood industry.The biopolymer must be deacetylated before chitosan can be produced.It can also be extracted using microbes in a biological extraction procedure.The development of products that take advantage of the bioactivities of the existing primary commercial source of chitin(crustacean)has lagged expectations.Also,the disadvantages of the present commercial source such as seasonality and competition for other uses among others has been one of the driving forces towards seeking alternative sources of chitin and chitosan in nature.This review highlights some of the efforts made by environmental scholars to locate possible commercial sources of chitin and chitosan in nature over time.

Effects of Chitin Nanocrystal on Solution Behavior and Gelation of Chitosan/β-Glycerophosphate Thermosensitive Hydrogel

The chitosan/β-glycerophosphate( CS/β-GP),a physical hydrogel system with thermosensitive and injectable features combined with biocompatibility and biodegradability, has great potentials as matrices for drug or cell encapsulation and delivery,or as in situ gel-forming materials for tissue repair. Here,the chitin nanocrystal( Chi NC) was introduced into the aforementioned system, and its effects on solution behavior and mechanical properties was investigated. The results showed the incorporation of Chi NC complicated sol-to-gel transition process; a higher loading ratio( 20%) speeded up sol-to-gel transition rate,reduced the solto-gel transition temperature,while still maintained shear-thinning behavior or injectable feature. Moreover,the mechanical properties of gels were significantly enhanced by Chi NC, accompanied by decreased water uptake. The above mentioned behavior favored better applications as injectable tissue-repair implants.

Optimization of the methods for extraction of chitin in crab shell and preparation of chitosan

A combination of both acid and alkali treatments was used to extract chitin from crab shell in this study. Then, a three factors (NaOH solution concentration, reaction time, reaction temperature) and three levels (35, 45, 55; 2, 6, 10; 70, 105, 140) L 9(3 4) orthogonal experiment design is further adopted to conduct a de acetyl treatment to prepare chitosan by considering the viscosity and de acetyl degree of the chitosan as the main performance indexes. Determination of de acetyl degree of chitin complys with the procedures given by the reference and the viscosity meter was used for determination of viscosity of chitosan. The results show that the extraction of chitin shall use pulverized crab shell as the raw material and such raw material shall be immersed in 10% HCl solution for 6 hours and washed with water for one time in every 2 hours, then heated in boiled water for 2 hours by the use of 10% thin NaOH solution. Afterwards, the said material shall be washed with water to become a neutral solution and dried over a stove. When chitin is mixed with 55% NaOH solution in a proportion of 1∶10 (W/V, g/mL) and the reaction takes place at a temperature of 105℃ for 6 hours, chitosan having a de acetyl percentage of 94% and viscosity of >200 cps can be available.

Evaluation of the Synergistic Effect of EDTA-Functionalized Chitosan Nanoparticles on Imipenem Delivery in Pseudomonas aeruginosa Carbapenem-Resistant Strain AG1

Metallo-β-lactamases are bacterial zinc-dependent enzymes involved in the hydrolysis of β-lactamic antibiotics representing the main cause of bacterial resistance to carbapenems, drugs of last resort for treating infections caused by multiresistant bacteria. We elaborated the hypothesis that it is possible to inhibit the enzymatic activity of metallo-β-lactamases by lowering the availability of zinc in the extracellular medium using metal chelating agents such as EDTA carried on nanoparticles. Chitosan, as linear cationic polysaccharide is frequently used in biomedical and pharmaceutical applications, has been studied as a biocompatible encapsulating agent in drug delivery systems and is an ideal transport agent for bioactive molecular complexes in antibiotic applications due to its ability to associate with negatively charged substances. We developed novel nanoparticles using chitosan as a transport matrix for β-lactamic antibiotics. Nanoparticles were synthesized according to the ion gelation method using tripolyphosphate as crosslinking agent. Nanoparticles were functionalized by the adsorption of EDTA, which acts as complexifying agent for Zn2+ ions causing inhibition of metallo-β-lactamases activity. We evaluate the antimicrobial effects of EDTA-functionalized nanoparticles with an imipenem cargo on the clinical isolate P. aeruginosa AG1, a carbapenem-resistant high-risk clone ST-111 carrying both blaIMP-18 and blaVIM-2 metallo-β-lactamases genes.

Anomalous Proton Conductivity in Chitin-Chitosan Mixed Compounds

In order to investigate a key factor for the appearance of proton conductivity in chitin-chitosan mixed compounds, the chitin-chitosan mixed compounds (chitin)x(chitosan)1-x were prepared and these proton conductivities have been investigated. DC proton conductivity σ is obtained from Nyquist plot of impedance measurement data, and the relationship between σ and mixing ratio x has been made clear. It was found that the x dependence of σ is non-monotonous. That is, σ shows the anomalous behavior, and has peaks around x = 0.4 and 0.75. This result indicates that there exist optimal conditions for the realization of high-proton conductivity in the chitin-chitosan mixed compound in which the number of acetyl groups is different. From the FT-IR measurement, we have found that the behavior of proton conductivity in (chitin)x(chitosan)1-x is determined by the amount of water content changed by x. Using these results, proton conductivity, which is important for the application of conducting polymers in chitin-chitosan mixed compounds, will be able to be easily controlled by adjusting the mixing ratio x.

β-Chitin/Chitosan Obtained from Loligo and Humboldt Squids Effectiveness as Wound Dressing Material

A number of materials are utilized to develop wound care dressing materials with metallic treatments such as ionic silver and zinc. Metallic ions if used for a prolonged time may lead to toxicity. Alternatively chitin,a natural polysaccharide found in nature, is utilized. It is found in fungi, crabs, mushrooms,squids, octopus, and many other living organisms. Chitin has similar structure to cellulose but its deacetylated derivate chitosan has amine groups that provide potential antibacterial properties along with a number of other advantages. Chitin in its natural form is found in three different structural forms,namely α,β,and γ.The β-chitin and chitosan are mostly found in the exoskeleton of squids. Loligo and Humboldt squids were studied. It is anticipated that Humboldt chitin is more effective in serving as antibacterial material and can be utilized for wound care. Differences in steriochemical structure were observed among β-chitin structures obtained and amine group's presences were found along with ability of materials to swell.

Extraction and Characterization of Litopenaeus vannamei’s Shell as Potential Sources of Chitosan Biopolymers

Chitin is the second most abundant polysaccharide,produced mainly as an industrial waste stream during crustacean processing.Chitin can be derived into chitosan through the deacetylation process.Conversion of shrimp waste into chitosan via the deacetylation process could be considered a practical approach for shell waste remediation.In this study,chitosan’s physicochemical characteristics extracted from two types of Pacific white leg shrimp,L.vannamei’s shell(i.e.,rough and smooth),were compared with commercial chitosan.The yield,moisture,ash,solubility,water and fat binding capacity were measured.The degree of deacetylation(DDA)was calculated using FTIR,and their chemical Structure was confirmed using XRD and SEM-EDS.Both extracted chitosan showed no significant difference in yield,moisture,ash,solubility and water binding capacity but showed a significant difference with commercial chitosan.Moreover,the fat binding capacity of commercial chitosan showed the lowest percentage(408.34±0.83%)as compared to extracted chitosan(smooth shell 549.59±12.48%;rough shell 500.55±12.10%).The DDA indicated that extracted chitosan from the smooth and rough shell was considered good chitosan as compared to commercial chitosan with 84.08±1.27%,80.78±0.79%and 74.99±1.48%,respectively.Additionally,the presence of hydroxyl and amino groups from FTIR and a good crystallinity index was recorded using XRD of extracted chitosan.Based on observed characteristics,shrimp shell waste from L.vannamei can achieve chitosan standard quality as a biopolymer and highly potential to be applied in various industrial applications.

Biocatalytic production of chitosan polymers from shrimp shells,using a recombinant enzyme produced by Pichia pastoris

Chitosan has a unique chemical structure with high charge density, reactive hydroxyl and amino groups, and extensive hydrogen bonding. Chitin deacetylase (EC 3.5.1.41) catalyzes the hydrolysis of the N-acetamido groups of N-acetyl-D-glucosamine residues in chitin, converting it to chitosan and releasing acetate. The entire ORF of the CDA2 gene encoding one of the two isoforms of chitin deacetylase from Saccharomyces cerevisiae was cloned in Pichia pastoris. The Tg (Cda2-6xHis)p was expressed at high levels as a soluble intracellular protein after induction of the recombinant yeast culture with methanol, and purified using nickel-nitrilotriacetic acid chelate affinity chromatography, resulting in a protein preparation with a purity of >98% and an overall yield of 79%. Chitin deacetylase activity was measured by a colorimetric method based on the O-phthalaldehyde reagent, which detects primary amines remaining in chitinous substrate after acetate release. The recombinant enzyme could deacetylate chitin, chitobiose, chitotriose and chitotetraose, with an optimum temperature of 50°C and pH 8.0, determined using oligochitosaccharides as the substrates. The recombinant protein was also able to deacetylate its solid natural substrate, shrimp chitin, to a limited extent, producing chitosan with a degree of acetylation (DA) of 89% as determined by Fourier transform infrared spectroscopy. The degree of deacetylation was increased by pre-hydrolysis of crystalline shrimp chitin by chitinases, which increased the deacetylation ratio triggered by chitin deacetylase, producing chito-oligosaccharides with a degree of acetylation of 33%. The results described here open the possibility to use the rCda2p, combined with chitinases, for biocatalytic conversion of chitin to chitosan with controlled degrees of deacetylation. We show herein that the crystalline chitin form can be cleanly produced in virtually quantitative yield if a combined and sequential enzyme treatment is performed.

Green Synthesis and Physical and Chemical Characterization of Chitosans with a High Degree of Deacetylation, Produced by a Binary Enzyme System

Chitosan is a biopolymer obtained from chitin, where the N-acetylglucosamine monomer is in its deacetylated form; this polymer is useful for a wide variety of industrial applications. The properties and uses of chitosan depend on its physical and chemical characteristics, which result from the treatments used for its production. In this study, we report the preparation and characterization ofchitosan oligosaccharides by a green synthesis from crystalline shrimp chitin, using a sequential enzyme treatment by chitinase and chitin deacetylase. Chitinases were purified from grapes and used to rupture the crystalline shrimp chitin structure, modifying the crystallinity index from 57.6% to 15.9%. The resultant polymers were deacetylated using a recombinant chitin deacetylase from Saccharomyces cerevisiae, which was cloned and expressed in Pichia pastoris. The chitosans produced showed an estimated DA （degree of acetylation） of approximately 20%, and the molecular weights ranged from -7,600 to -3,700 after treatment in pH 3.0 and pH 6.0 for 10 min and 40 min, respectively. Physical and chemical characterization of the products indicated that enzyme fragmentation of chitin probably makes the acetamide groups more accessible to deacetylation, forming homogeneous polymers that are free of hazardous sub-products, have defined low molecular weights, and are highly deacetylated.

An integrated theoretical and experimental investigation of insensitive munition compounds adsorption on cellulose,cellulose triacetate, chitin and chitosan surfaces

This manuscript reports results of combined computational chemistry and batch adsorption investigation of insensitive munition compounds, 2,4-dinitroanisole（DNAN）, triaminotrinitrobenzene（TATB）, 1,1-diamino-2,2-dinitroethene（FOX-7） and nitroguanidine（NQ）, and traditional munition compound 2,4,6-trinitrotoluene（TNT） on the surfaces of cellulose, cellulose triacetate, chitin and chitosan biopolymers. Cellulose,cellulose triacetate, chitin and chitosan were modeled as trimeric form of the linear chain of4 C1 chair conformation of β-D-glucopyranos, its triacetate form, β-N-acetylglucosamine and D-glucosamine, respectively, in the 1 ？ 4 linkage. Geometries were optimized at the M062 X functional level of the density functional theory（DFT） using the 6-31 G（d,p） basis set in the gas phase and in the bulk water solution using the conductor-like polarizable continuum model（CPCM） approach. The nature of potential energy surfaces of the optimized geometries were ascertained through the harmonic vibrational frequency analysis. The basis set superposition error（BSSE） corrected interaction energies were obtained using the 6-311 G（d,p）basis set at the same theoretical level. The computed BSSE in the gas phase was used to correct interaction energy in the bulk water solution. Computed and experimental results regarding the ability of considered surfaces in adsorbing the insensitive munitions compounds are discussed.

Treatment of fir wood with chitosan and polyethylene glycol

Chitosan is a natural biopolymer, derived from chitin, which is used for wood modification. Polyethylene glycol（PEG） was reacted with wood to provide possible fixation of the chitosan to wood. Wood blocks were treated with chitosan and PEG, as well as pre-treatment with the PEG at different temperatures and further reaction with the chitosan. The samples were soaked in water to study leaching of the chemicals, water absorption, swelling, as well as anti-swelling efficiency. Any prior reaction of the wood with PEG provided better reaction to the chitosan.Bulking was increased after the treatment of the wood with PEG. Swelling was reduced in the PEG-treated wood blocks as well as the pre-treated samples. Chitosan was not able to protect wood against water penetration： the treated samples showed more water absorption and swelling.However, pre-treatment of the samples decreased swelling in the wood, and the density was not noticeably affected by the treatments. Heating during the treatment caused more reduction in swelling for PEG–chitosan treated samples.

STUDIES ON CHITOSAN AND ITS pH-RESPONSIBILITY

This article is mainly concerned with speCial runctional membranes prepared from chitin, chitosan and surface poly(acrylic acid)-grarted chitin and their pHstimullus respensibility. The results showed that the Permeation or NaCl moleculesthrough the membranes were controlled by changing the pH or the membranes reyersiblyand the pH responsibility or chitosan membrane is quite oppposite or the surface poly(acrylic acid)-grarted chitin.

INFLUENCE OF MOLECULAR WEIGHT OF CHITIN AND ITS THREE DERIVATIVES ON CRITICAL CONCENTRATION OF LYOTROPIC LIQUID CRYSTALLINE PHASE TRANSITION

The critical concentration of lyotropic liquid crystalline phase transition for chitin derivatives was determined using a polarization microscope. The influence of molecular weight on critical concentration of liquid crystalline solution for chitin, chitosan, cyanoethyl chitosan and propionyl chitin successively increases as the chain rigidity decreases. Therefore it can be used as an indicator of the chain rigidity.

The Use of Exoskeletons of Shrimp(Litopenaeus vanammei)and Crab(Ucides cordatus)for the Extraction of Chitosan and Production of Nanomembrane

Chitin is widely distributed in nature, being the main structural component of the exoskeleton of crustaceans and is non-toxic, biodegradable and biocompatible. These exoskeletons once discarded become an industrial waste creating environmental pollutant. In order to find an alternative use, the present work exploits the extraction of the chitosan from chitin that is present in the exoskeletons of shrimps Litopenaeus vannamei and crabs Ucides cordatus and transforms it into high valued products, which can help solving the environmental problem as well to provide extra income to the fishermen. One example is the manufacture of nanomembranes from chitosan for the application in medical textiles. Nanomembranes using electrospinning of chitosan solutions (7% and 5wt%) with 100:0 v/v (TFA/DCM) and 70:30 v/v (TFA/DCM) were produced. Morphological properties of chitin and chitosan were studied using SEM, DRX, and thermal properties through TG/DTG and molecular structure by FTIR analysis. TG/DTG showed thermal decomposition of chitosan samples. X-ray diffraction analysis indicated the semi-crystalline structure of chitosan, and highly crystalline structure for chitin. Morphologies of the nanomembranes were also observed from scanning electron micrographs. Results showed that the nanomembranes with 5% chitosan solutions with 70:30 v/v (TFA/DCM) showed facilitation in the formation of the nanomembranes. The nanomembranes of shrimp and crab with 5% 70:30 v/v (TFA/DCM) had higher breaking tension and breaking extension. With positive results obtained, the present work will help the authorities to organize the fishermen to have consciousness in the collection of exoskeleton waste as well as helping to have a better environment.

Preparation of Two Different Serials of Chitosan

Two parts of research work on preparation of chitosan are presented. One is about the optimization of preparation condition and the other about the preparation of two series of chitosan with special structures. The first series has the same degree of deacetylation but different molecular weights; and the second the same molecular weight but different in degree of deacetylation.

Potential Economic Value of Chitin and Its Derivatives as Major Biomaterials of Seafood Waste,with Particular Reference to Southeast Asia

With a growing population,changes in consumerism behavior and trends in consumption in Indo-Pacific Asia,our seafood processing and consumption practices produce a large volume of waste products.There are several advantages in regulating and sustaining shellfish processing industries.The major advantage of waste management is that it leads to better conservation of natural resources in the long run.Shrimp shell waste contains useful biomaterials,which are still untapped due to inadequate waste disposal and solid waste management.Chitin,the major component of shell waste,can be extracted either chemically or biologically.The chemical extraction approaches,which use acids and alkali,could be an environmental burden.On the other hand,biological methods can be eco-friendly alternatives for shell waste management.In this review,recent trends in management of shellfish waste as sources of chitin,conversion of chitin into chitosan,economic aspects of waste treatment and application of chitosan will be discussed.

Impact of pH Variation on Coag-flocculation Behaviour of Chitin Derived Coag-flocculant in Coal Washery Effluent Medium

This work investigates the influence of pH variation on coag-flocculation kinetics and performance of Chitin Derived Coag-flocculant (CDC) in removal of Suspended and Dissolved Particles (SDP) from Coal washery effluent (CWE) medium. Key parameters such as rate constant Km, half life t1/2,, and pH etc. were investigated. The best coag-flocculation performance is recorded at Km, of 0.007 l/mg.min, t1/2, of 0.0362min, pH of 8, dosage of 100mg/l and efficiency E(%) of 99.933. Minimum efficiency (%) > 94.00 was achieved at 30 minutes of coagflocculation, establishing CDC as an effective water treatment agent at the conditions of the experiment.

Two Routes to Produce Chitosan from Agaricus bisporus

Two methods were used to produce chitosan by deacetylation of chitin which was extracted from Agaricus bisporus stipes.The first one gives chitosan 1 with low yield of 2.5%,degree of acetylation(DA)of 4%,molecular weight(MW)of 2.973×10^(5)(g/mol).The second route produces chitosan 2 with higher yield of 41%,degree of acetylation(DA)of 17.23%,molecular weight(MW)of 2.939×10^(5)(g/mol).Both chitosans were characterized by XRD,FTIR,^(1)H-NMR spectroscopy nuclear magnetic resonance of proton.The molecular weight(MW)was determined by size exclusion chromatography(SEC).Thermal analysis shows that both chitosans have moisture content lower than 10%.However chitosan 2 has the less ash%which is the quality grade for chitosan medical applications.Accordingly,fungal chitosan 2 could have potential medical and agricultural applications.

Extraction and Characterization of Chitosan from Shrimp Shells

Chitosan is one of the most studied polysaccharides nowadays. Because it is non-toxic, widely used in food, pharmaceutical processes, agricultural, and presents excellent biological properties such as biodegradation in the human body, and antibacterial. In the present study we reported the extraction of low cost chitosans (Cs1, Cs2, Cs3 and Cs4) from shrimp shells by extraction of chitin (Egypt: case study), then alkaline deacetylation of chitin with strong alkaline solution at different period of time. The different prepared chitosans (Cs1, Cs2, Cs3 and Cs4) were characterized by FTIR spectroscopy, thermal stability, morphology, crystallography, elemental analysis and degree of deacetylation. The data showed that the prepared chitosan Cs2 has the most thermal stability and the highest degree of deacetylation.