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.

Biological Properties of Chitosan Films with Different Degree of Deacetylation

Chitin and chitosan films were prepared by solution casting method. Chitosan specimens used in this study were deacetylated by 50.4%, 69.2%, 85.5% and 96.3%. Their water content, protein adhesion ability, cytocompatibility, cell adhesion ability, in vitro and vivo degradability and biocompatibility were evaluated. Results indicated that with the degree of deacetylation （DD） between 50% and 70%, the chitosan showed higher water content. The higher the DD, the stronger protein adhesion ability the chitosan had. All the films have good cytocompatibility and the films with higher DD have better cell adhesion ability. Chitin films degraded more rapidly than others, which disappeared in 2 to 4 weeks after they were implanted in subcutaneous tissue and musculature. Their inflammatory reaction became weaker as the films degraded. As the DD got higher, the films degraded slower. The films of DD 85.5% and DD 90.3% even didn＇t disappeared in 12 weeks after they were implanted. Their inflammatory reaction was mild at the beginning of degradation, and became severe in 4 to 8 weeks, then weaken at last. This basic result can be very helpful for tissue engineering.

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.

Immunostimulatory eff ect of quaternary degree and acetyl group of quaternized chitosan on macrophages RAW 264.7

Hydroxypropyltrimethyl ammonium chloride chitosan(HACC)and hydroxypropyltrimethyl ammonium chloride fully deacetylated chitosan(De-HACC)were synthesized with various degrees of substitution by altering the ratio of chitosan to glycidyl trimethyl-ammonium chloride(GTMAC).The effects of the quaternary ammonium degree and the acetyl group of these polymers on immunostimulatory activities were detected in RAW 264.7 cells.The expression levels of nitrogen oxide(NO),interleukin-6(IL-6)and tumor necrosis factor(TNF-α)were compared.Results show that the removal of acetyl groups in chitosan obviously improved the degree of substitution of quaternary ammonium salts.In addition,HACC and De-HACC were capable of promoting immunological activity in a substitution-dependent manner;HACC was positively correlated,and De-HACC was negatively correlated.Among tested ratios,HACC-30%and De-HACC-54%performed better than the others,and De-HACC-54%performed the best.Generally,quaternized chitosan possesses immunostimulatory activity,which is related to the degree of quaternization and the acetyl group.

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.

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.

Chitosan Obtained from Cell Wall of Aspergillus Niger Mycelium

Chitin from cell walls of Aspergillus Niger mycelium was prepared. A new method for the preparation of high deacetylation degree chitosan was studied in a dilute sodium hydroxide solution at a high pressure. The experimental results indicate that the deacetylation degree of the chitosan can reach 80% under the condition of a 5.00 mol/L sodium hydroxide solution at 0.1 MPa of pressure for 1 h. This method shows the advantages of the applications in the industry production and environment protection.

Determination of degree of substitution for N-acylated chitosan using IR spectra

Chitosans with various degrees of deacetylation (D.D.), which were used as standard sample for FTIR determination, were prepared from completely deacetylated chitosan by homogeneous N-acetylation reaction. By combining four probable probe bands, i.e. 1655, 1560, 1380 and 1320 cm-1, eight probable reference bands, i.e. 3430, 2920, 2880, 1425, 1155, 1070, 1030 and 895 cm-1 and two baseline methods, the most suitable ratios Aprobe band/Areference band from IR spectra to determine the degree of acetylation of chitosan were evaluated from 48 combinations to be A1560/A2880, A1560/A2920 and A1655/A3430(A1560/A2880 is mostly recommended). The second baseline method, i.e. linking between adjacent two valleys, was better for measuring the absorbances of 1560 and 1655 cm-1 bands. The determination range of the D.D. (1%-100%) covered almost the whole range. The standard curves with A1560/A2880 and A1655/A3430 were also suitable for the determination of degree of substitution of other N-acylated chitosan, such as N-propionyl chitosan, N-butyryl chitosan and N-hexanoyl chitosan.

Technology about preparation for chitosan reserving protein

采用D -近似最优设计法系统地研究了NaOH质量分数、碱处理时间及碱处理温度这三个主要因素对制备壳聚糖的影响 :当碱液质量分数增加时 ,壳聚糖的脱乙酰度增加 ,但其速度却在减小 ,当碱液质量分数达到 40 %时 ,脱乙酰度出现峰值 ,约为 90 % ,而后增加碱液质量分数时 ,脱乙酰度反而下降 ;随着反应温度的升高 ,其脱乙酰度几乎线性递增 ,当温度达到 2 0 0℃附近时 ,曲线趋于平直并且脱乙酰度达到最大 ;随着反应时间的增加 ,脱乙酰度开始呈线性增加 ,当反应时间超过 5 0min后 ,脱乙酰度有下降趋势 ;在一定碱液质量分数 (4 0 % )条件下 ,脱乙酰度随着温度的增加而增加 ,因此 ,若需获得较高质量的壳聚糖 。

Bone remodeling effect of a chitosan and calcium phosphate-based composite

Chitosan is a biocompatible polymer that has been widely studied for tissue engineering purposes.The aim of this research was to assess bone regenerative properties of an injectable chitosan and calcium phosphate-based composite and identify optimal degree of deacetylation(%DDA)of the chitosan polymer.Drill holes were generated on the left side of a mandible in Sprague-Dawley rats,and the hole was either left empty or filled with the implant.The animals were sacrificed at several time points after surgery(7–22 days)and bone was investigated using micro-CT and histology.No significant new bone formation was observed in the implants themselves at any time points.However,substantial new bone formation was observed in the rat mandible further away from the drill hole.Morphological changes indicating bone formation were found in specimens explanted on Day 7 in animals that received implant.Similar bone formation pattern was seen in control animals with an empty drill hole at later time points but not to the same extent.A second experiment was performed to examine if the%DDA of the chitosan polymer influenced the bone remodeling response.The results suggest that chitosan polymers with%DDA between 50 and 70%enhance the natural bone remodeling mechanism.

Rhizopus stolonifer几丁质脱乙酰酶的异源表达及其与几丁质酶的协同作用研究

几丁质脱乙酰酶(chitin deacetylase,CDA)是几丁质生物转化为壳聚糖的关键酶。然而,目前报道的高活性CDAs非常少。为了获得高效的CDA,并提高其对几丁质的生物转化效率,该研究对来源于Rhizopus stolonifer中的CDA进行基因克隆、表达及纯化,获得几丁质脱乙酰酶Rs CDA1,研究其酶学性质并探究其与来源于Vibrio harveyi的几丁质酶Vh Chit2对几丁质的协同水解作用。结果表明,Rs CDA1的比酶活力为5.2 U/mg,在45℃、pH 6.5条件下活性最高,在温度低于40℃、pH 6.5~8.0保持较好的稳定性。1 mmol/L的Ca^(2+)、Mn^(2+)、Mg^(2+)、Zn^(2+)、Ba^(2+)和Co^(2+)对Rs CDA1的活力有促进作用;1 mmol/L的Ni^(2+)和Cu^(2+)对该酶有抑制作用。此外,Rs CDA1与Vh Chit2在几丁质水解中表现出良好的协同降解和脱乙酰作用。Rs CDA1与Vh Chit2(摩尔浓度比为10∶1)在50℃、pH 6.5条件下水解20 mg/mL几丁质2 h时,对几丁质酶和CDA的协同度分别达到120.3%和175.6%。与单独使用Rs CDA1相比(27.5%),两者协同水解时几丁质的脱乙酰度增加到35.3%。因此,Rs CDA1在几丁质的生物转化中具有潜在的应用前景。

羧甲基壳聚糖脱乙酰度和取代度检测技术综述

随着羧甲基壳聚糖作为生物材料在医疗器械行业中被广泛应用,迫切地需要能够有效测定其脱乙酰度和取代度的方法。与此同时,存在着羧甲基壳聚糖脱乙酰度和取代度的检测技术不完善,检测方法欠缺等问题。因此,该文简述了灰分分析法、胶体滴定法、核磁共振法、傅里叶变换红外光谱法、电位滴定法、电导滴定法、顶空气相色谱法检测羧甲基壳聚糖脱乙酰度和取代度的原理、方法,分析讨论了各方法的优缺点。最后,展望顶空气相色谱检测技术对羧甲基壳聚糖的脱乙酰度和取代度的研究前景,随着该方法研究的深入和广泛开展,可以进一步促进羧甲基壳聚糖质量评价技术的发展。

不同特性壳聚糖对法兰克福香肠品质的影响

为获得一种具有优良性能和健康功效的新型乳化类肉制品,本研究评价了不同分子量(3.2~676 kDa)和不同脱乙酰度(70%~90%)的壳聚糖对法兰克福香肠品质特性的影响。结果表明,分子量670 kDa、脱乙酰度90%的壳聚糖具有最好的增稠能力,在HMHD添加量为1.0%时可显著提高香肠的a^(*)值、硬度、内聚力和pH值,同时可以降低蒸煮损失率(P<0.05)。因此,分子量670 k Da、脱乙酰度90%的壳聚糖可以更好地改善香肠的品质。本研究可为获得具有优良性能和健康功效的新型功能性肉制品提供理论支持。

从营口对虾虾壳制备壳聚糖的研究

以营口本地对虾虾壳为原料,用稀HCI和NaOH溶液分别脱去钙和蛋白质及脂肪得到甲壳素样品,使用浓碱加热处理得到壳聚糖。通正交试验设计计优化了壳聚糖的制备过程。结果表明壳聚糖制备的最佳条件是先用稀酸脱盐,再用稀碱脱蛋白得到甲壳素,随后用45%NaOH溶液,在95℃、反应时间6 h下得到壳聚糖。自制壳聚糖与市售壳聚糖的红外光谱图一致。用酸碱滴定法和红外光谱法分别测定了样品的脱乙酰度。重量法测定壳聚糖的水分。乌氏粘度计测定壳聚糖大的平均相对分子质量。所得壳聚糖的水分含量为14%,脱乙酰度为74%,平均相对分子质量为4.30×10^(4)。

响应面法优化制备高脱乙酰度小龙虾壳聚糖工艺及产物表征

以小龙虾虾壳为原料,脱乙酰度(DD)为评价指标,响应面法优化制备高脱乙酰度壳聚糖工艺。结果表明,最佳制备工艺为:氢氧化钠浓度50%、球磨处理时间12 h、料液比1:15(g/mL),在此条件下进行验证试验,可得到脱乙酰度为88.1%的小龙虾壳聚糖。制备得到的高脱乙酰度小龙虾壳聚糖颗粒均匀有序,适合作为壳聚糖解聚原料,具有广泛应用前景。为小龙虾副产物开发利用提供理论参考。

NMR、FTIR及电位滴定法测定壳聚糖脱乙酰度

脱乙酰度是影响甲壳素、壳聚糖及其衍生物物化特性和生物活性功能的重要参数。介绍了三种壳聚糖脱乙酰度的测定方法。以13C交叉极化魔角旋转固体核磁法测定的结果作为参照,对红外光谱法和线性电位滴定法的测定结果进行了分析比较,并分析了不同测定方法的误差来源。

碱量法测定壳聚糖脱乙酰度计算公式中存在的一个问题的探讨

碱量法(包括酸碱滴定法和电位滴定法)是目前测定壳聚糖脱乙酰度较为常用的方法,其原理是用过量的稀酸与一定量的待测壳聚糖反应,然后用标准 NaOH 溶液滴定过量的酸来测量壳聚糖中自由氨基的量,从而计算壳聚糖的脱乙酰度(DD 值)。计算公式多为:DD = (NH2% / 0.0994) * 100%。本文分析了这一计算公式中存在的问题,并提出了相对正确的计算公式。

不同来源壳聚糖的基本特性及红外光谱研究

用同一方法在相同条件下，分别以螯虾壳、河虾壳、对虾壳、海蟹壳和蚕蛹制备了壳聚糖，并对其多项特性进行了比较研究。壳聚糖的收率分别为螯虾１３．６％、海蟹１２．３％、对虾１０．５％、河虾８．７０％、蚕蛹１．８％；灰份含量接近（０．２５～０．４５％）；脱乙酰度相近（７６～８０％）；壳聚糖的粘均分子量分别为：螯虾１２．８×１０６、河虾１．１５×１０６、海蟹１．０６×１０６、对虾０．９６×１０６、蚕蛹０．９１×１０６。五种壳聚糖的红外光谱基本相似，只存在个别峰值、峰位的差异，表明五种壳聚糖的基本结构相同，但其空间构象和结晶度等方面存在差异。

甲壳素微波法脱乙酰制备壳聚糖的研究

本文采用微波技术由甲壳素脱乙酰制备壳聚糖,首次对在微波反应前采用乙醇浸泡对甲壳素进行预处理的条件进行了较为系统的研究,并通过单因素试验和正交试验分析了微波反应时间、碱液浓度、乙醇浸泡时间、乙醇浓度、微波功率对壳聚糖脱乙酰度和粘均分子量的影响,提出了制备高脱乙酰度、高粘均分子量壳聚糖的最优条件为(微波功率462W):微波反应时间20min,NaOH浓度50%,乙醇浸泡时间2.5h,乙醇浓度80%。在该条件下制得的壳聚糖脱乙酰度为77.07%,粘均分子量为43.13万,得率为68.98%。

天然生物材料——甲壳素和壳聚糖的开发及应用

甲壳素／壳聚糖是非常有价值的天然生物材料，对它的开发和利用有着广阔的前景。本文综述了甲壳素／壳聚糖的结构与性能、制备与表征方法以及它们在医药、工业和农业等方面的应用。