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.

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.

Preparation and Application of Chitosan-based Polyelectrolyte Complex Materials: An Overview

Chitosan,a renewable,non-toxic,and natural cationic polyelectrolyte,can be combined with many anionic polyelectrolytes(such as sodium alginate,hyaluronic acid,xylan,and gelatin)via electrostatic forces to form chitosan-based polyelectrolyte composites under certain conditions.This review summarizes various methods of preparing chitosan-based polyelectrolyte composites and analyzes their applications in clinical medicine and agriculture,as well as pharmaceutical,tissue,food,environmental,and textile engineering fields.The future development direction and potential of chitosan-based polyelectrolytes are also discussed.

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.

Chitin oligosaccharides for the food industry:production and applications

Chitin oligosaccharides(CHOS),high-value-added oligomers linked by N-acetyl-d-glucosamine(GlcNAc,NAG),and a small amount of d-glucosamine(GlcN,GA),have aroused increasing interest due to their excellent biological properties,including antibacterial,anti-inflammatory,and immunoprotective activities,and intestinal regulation.The efficient production and utilization of CHOS with high performance can solve problems from chitin as biowaste.However,the large-scale production of well-defined CHOS has not been fully accomplished due to the limited biotechnology and separation methods,thus impeding the research on their biological functions as well as their accurate applications.In this review,we comprehensively summarize the current preparation methods of CHOS,including the chemical,physical,enzymatic and biosynthetic methods.The advantages and disadvantages of the methods are discussed in terms of efficiency,economy,and environmental effects.Furthermore,the applications of CHOS in the food industry and their contributions to human health based on their excellent bioactivities are expounded.It is hoped that this review will help in providing new insights into the production of CHOS with high precision,and support the application of CHOS in serving the food industry as nutritional supplements or foods for special medical purposes.

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.

Preparation of magnetic chitosan microspheres and its applications in wastewater treatment

The methods of preparation of magnetic chitosan microspheres have been introduced. In addition, their applications in the wastewater treatment, based on different kinds of wastewater, have been reviewed, and their mechanisms have been discussed.

Preparation of Chitin-PLA laminated composite for implantable application

The present study explores the possibilities of using locally available inexpensive waste prawn shell derived chitin reinforced and bioabsorbable polylactic acid(PLA)laminated composites to develop new materials with excellent mechanical and thermal properties for implantable application such as in bone or dental implant.Chitin at different concentration(1e20%of PLA)reinforced PLA films(CTP)were fabricated by solvent casting process and laminated chitin-PLA composites(LCTP)were prepared by laminating PLA film(obtained by hot press method)with CTP also by hot press method at 160
C.The effect of variation of chitin concentration on the resulting laminated composite's behavior was investigated.The detailed physico-mechanical,surface morphology and thermal were assessed with different characterization technique such as FT-IR,XRD,SEM and TGA.The FTIR spectra showed the characteristic peaks for chitin and PLA in the composites.SEM images showed an excellent dispersion of chitin in the films and composites.Thermogravimetric analysis(TGA)showed that the complete degradation of chitin,PLA film,5%chitin reinforced PLA film(CTP2)and LCTP are 98%,95%,87%and 98%respectively at temperature of 500
C.The tensile strength of the LCTP was found 25.09 MPa which is significantly higher than pure PLA film(18.55 MPa)and CTP2 film(8.83 MPa).After lamination of pure PLA and CTP2 film,the composite(LCTP)yielded 0.265e1.061%water absorption from 30 min to 24 h immerse in water that is much lower than PLA and CTP.The increased mechanical properties of the laminated films with the increase of chitin content indicated good dispersion of chitin into PLA and strong interfacial actions between the polymer and chitin.The improvement of mechanical properties and the results of antimicrobial and cytotoxicity of the composites also evaluated and revealed the composite would be a suitable candidate for implant application in biomedical sector.

Progress and prospects in chitosan derivatives:Modification strategies and medical applications

Chitosan(CS)is the only natural alkaline polysaccharide originated from deacetylation of chitin that is the main component of shell from marine organisms.It has great potential medical application due to its broad-spectrum antimicrobial activity and good water solubility originated from its protonated amino groups under acidic condition and abundant hydroxyl groups.However,unprotonated NH_(2)group of CS leads to its poor solubility under physiological condition and limits its diverse applications.Therefore,it is highly necessary to summarize the modification strategies of CS derivatives systematically to help researchers select the most appropriate strategies for their specific applications.Herein,we have summarized the modification strategies of CS derivatives for improving their antimicrobial activity,water solubility,biocompatibility,and mechanical property by chemical reaction and physical integration.And then we have reviewed the CS derivatives in hydrogels,nanoparticles,or coatings for medical application in wound dressing,drug delivery,medical implant.Last but not the least,we have put forward the future perspectives of deep studies about structure-activity relationship and clinical applications of CS derivatives.

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.

Sources,production and commercial applications of fungal chitosan:A review

Chitosan is a type of biopolymer that can be obtained from animal/marine sources,and it can also be extracted or produced from agriculture waste products like mushroom or different fungal sources after the chitin deacetylation.Depending on the size of mushroom farm,the amount of waste ranges between 5%and 20%of the production volume.The cell wall of the filamentous fungi,a good source of chitin,offers an easy way to extract chitin.The physicochemical character-istics such as molecular weight and degree of deacetylation of fungal chitosan can be controlled compared to chitosan obtained from crustacean sources.Fungal sourced chitosan can be used in food,pharmaceutical or biomedical applications for different applications,for example,as an antimicrobial agent,coating material,water purification or bio-pesticide.This review mainly fo-cused on the extraction of chitin from mushroom or different fungal sources and also showed some applications of commercial chitosan products.

Properties of Microcrystalline Chitosan-Calcium Phosphate Complex Composite

Nature itself uses materials like, cellulose to provide the structure of plants, chitin as the exoskeleton of several insects and molluscs, collagen for mechanical support in connective tissues and so on. At present, the socioeconomic situation of the modern world has raised the interest in renewable materials being used in regenerative medicine. The composition of MCCh/?-TCP complex in sponge shape is derived from the junction of two or more different materials, containing organic and inorganic materials, including bioactivity and biodegradability as a characteristic. The chemical characteristics of MCCh/?-TCP complex composites showed that both of the components organic and inorganic exist in the material. All sponge preparations, with MCCh/?-TCP have a well-shaped 3-dimentional structure, a highly porous and interconnected and homogenous pore structure to ensure a biological environment conducive to cell attachment and proliferation as well as tissue growth, providing the passage of nutrient flow. These materials can be used in future for medical applications as a base for scaffolds production and as implants in regenerative medicine.

Zingiber cassumunar blended patches for skin application:Formulation,physicochemical properties,and in vitro studies

Our work was to study the preparation,physicochemical characterization,and in vitro characteristic of Zingiber cassumunar blended patches.The Z.cassumunar blended patches incorporating Z.cassumunar Roxb.also known as Plai were prepared from chitosan and polyvinyl alcohol with glycerin as plasticizer.They were prepared by adding all ingredients in a beaker and homogeneously mixing them.Then,they were transferred into Petri-dish and dried in hot air oven.The hydrophilic nature of the Z.cassumunar blended patches was confirmed by the moisture uptake,swelling ratio,erosion,and porosity values.The FTIR,DSC,XRD,and SEM studies showed revealed blended patches with amorphous region that was homogeneously smooth and compact in both surface and cross section dimensions.They exhibited controlled the release behavior of(E)-4-(30,40-dimethoxyphenyl)but-3-en-lol(compound D)that is the main active compound in Z.cassumunar for anti-inflammation activity.However,in in vitro skin permeation study,the compound D was accumulated in newborn pig skin more than in the receptor medium.Thus,the blended patches showed the suitable entrapment and controlled release of compound D.Accordingly,we have demonstrated that such chitosan and polyvinyl alcohol formulated patches might be developed for medical use.

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.