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.

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.

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.

The Evaluation on Biological Properties of Carboxymethyl-chitosan and Carboxymethyl-chitin

Carboxymethyl-chitosan and carboxymethyl-chitin were prepared with the methods developed in our laboratory. The DS (degree of substitution) and DD (degree of deacetylation) of the carboxymethyl-chitosan were 103.14% and 97.18% respectively,while the DS of the carboxymethyl-chitin was 96.37%. Their effects on human fibroblasts,intradermal irritation test,in vitro and vivo degradability,and biocompatibility were evaluated. The results indicate that the polysaccharides at low concentrations can facilitate the growth of human fibroblasts and the carboxymethyl-chitosan at 100 μg mL-1 is the most effective. The polysaccharides at higher concentrations,however,inhibit the growth of fibroblasts. The PII (Primary Irritation Index) values of CM-chitosan and CM-chitin are both 0.0,which shows that they have no irritation reaction. Both of the polysaccharides show good degradability and biocompati-bility. Carboxymethyl-chitin degrades faster in vitro than carboxymethyl-chitosan. The latter,however,has no inflammatory reaction after being implanted in vivo for 7 d and shows better biocompatibility. This study may provide a scientific basis for the use of car-boxymethyl-chitosan and carboxymethyl-chitin as biomaterials.

新型缓释EDTA对土壤铅镉释放的影响

利用羧甲基壳聚糖的环境友好性和成模性将其作为缓释载体,将EDTA和羧甲基壳聚糖按照1∶1的配比通过喷雾干燥器制备了的缓释EDTA。以缓释EDTA为研究对象,进行了表征分析、缓释动力学研究、土壤重金属静态活化特性和动态淋溶特性研究。结果表明,缓释EDTA包封率可达87.51%,在水中释放过程符合Higuchi模型,具有明显的缓释效果。缓释EDTA相比EDTA可以在提高土壤溶液中Pb、Cd离子浓度的同时有效控制Pb、Cd离子的突增,促使土壤溶液中的Pb、Cd离子保持适量浓度持续提升。动态淋溶结果表明,添加两种不同形式的EDTA会显著提升Pb、Cd的淋失量,但缓释EDTA处理能显著降低Pb、Cd的初始淋失量和淋失总量,其中Pb和Cd的淋失总量分别降低了19.5%和20.9%。

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.

EDTA脱钙法制备甲壳素

The methods using EDTA and HCl to remove CaCO3 in preparation of Chitin were studied by IR spectrometer and analyzing the properties of chitin.The results showed that EDTA could replace HCl in the production of chitin for removing CaCO3.Especially owing to the mechanism of removing CaCO3 that EDTA possessed,there was nearly no damage to the molecular chains of chitin during the process of removing CaCO3.The molecule mass of its product was bigger than that product treated by HCl for removing CaCO3.Therefore,The high relative molecular mass chitin and chitosan could be prepared in the same condition,and all the other properties such as productivity,degree of deacetylation and color were better than that product treated by HCl.Furthermore,this process could reduce the pollution in production of chitin,and the cost was not higher than the process with HCl.

壳聚糖和壳聚糖EDTA接合物双层包覆胰岛素口服纳米脂质体的研究

目的 研究壳聚糖和壳聚糖 EDTA接合物 (CEC)双层包覆胰岛素脂质体的性质、降血糖作用和药代动力学。方法 采用逆相蒸发法制备胰岛素脂质体 ;用胃蛋白酶、胰蛋白酶溶液和胃肠道内容物试验考察脂质体对胰岛素的保护作用 ;用酶 苯酚法测定小鼠血糖值 ;用放射免疫法测定血清胰岛素含量 ,并采用Pkanalyst程序进行拟合。结果 在胃蛋白酶、胰蛋白酶和胃肠道内容物中 ,壳聚糖 CEC双层包覆胰岛素脂质体对胰岛素具有较好的保护作用 ;在正常大鼠葡萄糖耐药量试验中 ,与PBS对照组比较 ,壳聚糖及其CEC包覆胰岛素脂质体对负载葡萄糖大鼠的血糖升高均有一定的抑制作用 ,其中壳聚糖 CEC双层包覆胰岛素脂质体的抑制作用最佳 ;在大鼠降血糖试验中 ,壳聚糖及其CEC包覆胰岛素脂质体均具有一定降血糖作用 ,以壳聚糖 CEC双层包覆胰岛素脂质体的降血糖作用最佳 ,血糖在 1h降至最初血糖值的 4 5 98% ,作用时间延长 ;以皮下注射胰岛素 (Ins)为对照 ,其相对生物利用度为17 0 2 % ;对血清Ins浓度 时间曲线进行拟合计算 ,均符合一室线性模型 ,以皮下注射Ins为对照 ,壳聚糖 CEC双层包覆胰岛素脂质体的相对生物利用度为 8 91%。结论 采用壳聚糖 CEC双层包覆的胰岛素脂质体更有利于胰岛素口服吸收。

制备甲壳素过程中EDTA脱钙法的研究

通过对不同条件下制备而成的甲壳素进行灰分(含钙量)、红外光谱、扫描电镜等分析测试,比较了采用EDTA溶液代替盐酸制备甲壳素的脱钙效果以及甲壳素在结构、性能参数等方面的变化。其结果为:无论是用pH=4,还是pH=10的EDTA饱和溶液,其脱钙效果都远远大于pH=4的盐酸溶液;由不同脱钙法所制得的甲壳素其结构基本一致,但相对分子质量有较大差别,其中以EDTA脱钙法制备的甲壳素相对分子质量较大。

超声波辅助EDTA法提取淡水小龙虾壳中甲壳素的工艺研究

以烹饪前后淡水小龙虾壳为原料,研究超声辅助EDTA法提取淡水小龙虾壳中甲壳素的最佳工艺条件,采用EDTA法提取甲壳素,以提取物的灰分含量为评价指标,通过单因素试验和正交试验选择最佳的EDTA浓度、料液比、反应p H值、反应时间,结果表明:EDTA法提取甲壳素的最佳条件为,料液比1∶26(W/V)、EDTA浓度18%、反应时间2.5 h、反应p H值13。在此最佳提取条件下辅助超声波法提取甲壳素,以提取物灰分含量和含氮量作为提取效果的评价指标,筛选最佳超声时间、料液比和超声处理温度,结果表明:超声波辅助EDTA提取甲壳素的最佳工艺条件为处理温度30℃、p H值13、EDTA浓度18%、料液比1∶24(W/V)、超声处理时间45 min、超声频率60k Hz、功率180 W。在此条件下,烹饪后甲壳素提取物灰分含量和含氮量均较烹饪前低,分别为0.13%和3.51%。

壳聚糖和EDTA对污染土壤中Pb的解吸作用研究

通过吸附-解吸试验分别研究了壳聚糖和EDTA对污染棕红壤土中Pb的解吸作用。结果表明,壳聚糖螯合剂及EDTA均能显著提高棕红壤中吸附态铅的解吸效率,增加解吸溶液中Pb的浓度。壳聚糖对污染土壤中Pb的解吸作用随着壳聚糖加入量的增大而增大,在达到一定浓度时提取率达到最高值,最后趋向平缓,而提取液pH值的变化对提取率影响较小;EDTA的提取率随其加入量增大而增加,然后趋缓,而提取液pH的增加提取率则呈先下降后上升的趋势,在pH为3时最低。壳聚糖与EDTA比较而言,两者的提取率比较接近。

壳聚糖-EDTA轭合物对胰岛素口服纳米脂质体的影响

目的 :研究不同修饰程度和不同浓度的壳聚糖 EDTA轭合物 (CEC)包覆胰岛素脂质体的性质和口服降血糖作用。方法 :用壳聚糖合成CEC ;采用逆相蒸发法制备胰岛素脂质体 ,用CEC对胰岛素脂质体进行包覆 ;用透射电镜和光子相关光谱及激光多谱勒测定仪测定其形态、Zeta电位和粒径 ;用HPLC法和超速离心法测定包封率 ;用胃蛋白酶和胰蛋白酶溶液实验考察CEC包覆脂质体对胰岛素的保护作用 ;用酶 -苯酚法测定小鼠血糖值。结果 :当壳聚糖的D 葡糖胺亚单位与EDTA的摩尔比为 1:10时 ,CEC的粘度最高。在CEC包覆胰岛素脂质体中 ,随着修饰度的增加 ,Zeta电位和粒径稍有下降 ;随CEC浓度增加 ,Zeta电位和粒径则相应有所增加。包封率与CEC修饰度和浓度相关性不大 ,形态均为球形或近球形。在胃蛋白酶溶液中 ,CEC对胰岛素溶液具有较好的保护作用 ,用CEC包覆胰岛素脂质体对胰岛素具有明显的保护作用 ,其中以CEC2的保护作用最佳 ,且随浓度的增加而有所增强。在胰蛋白酶溶液中 ,CEC本身对胰岛素溶液有较好的保护作用 ;但用CEC包覆的胰岛素脂质体反而加速胰岛素的降解。CEC包覆的胰岛素脂质体均具有一定的降血糖作用 ,以CEC2包覆的胰岛素脂质体降血糖作用最佳 ,并呈浓度依赖性 ,其中 2

壳聚糖修饰玻碳电极卷积伏安法测定环境水中的EDTA

制备了壳聚糖修饰玻碳电极 ,研究了Fe(EDTA) -在修饰电极上的吸附还原行为 ,用卷积伏安法通过Fe(EDTA) -的检测测定了环境水中的EDTA。在优化的实验条件下 ,峰电流值与 8.0× 1 0 -7～ 5 .0× 1 0 -6mol L的EDTA呈线性关系 ,回归方程为epp′ =0 .75 2 5c - 0 .661 3,r =0 .991 ,最低检出限为 5 .0× 1 0 -7mol L ,5次测定的相对标准偏差小于 5 .8%。对实际样品测定的回收率为98 1 %～ 1 0 5 % ,对比HPLC的结果 ,相对偏差小于 5 %。

EDTA对壳聚糖的抑菌增效研究

为了研究壳聚糖与EDTA的协同抗菌效果,采用体外药敏片扩散法和抑菌效应方法,测试了壳聚糖和EDTA联合应用后对大肠杆菌、金黄色葡萄球菌、白色念珠菌的抑制效应,来考察EDTA对壳聚糖的抑菌增强效果。结果表明,与壳聚糖对照组相比,壳聚糖和EDTA联合应用能增强壳聚糖对白色念珠菌、金黄色葡萄球菌、大肠杆菌的抑制杀伤作用,且对大肠杆菌的抑制杀伤效应更加明显。表明EDTA能够增强壳聚糖对细菌、真菌的抑制效应,为开发一种新型的抗菌剂提供了试验依据。

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.