Crab-Eating Monkey Acidic Chitinase (CHIA) Efficiently Degrades Chitin and Chitosan under Acidic and High-Temperature Conditions

Chitooligosaccharides, the degradation products of chitin and chitosan, possess anti-bacterial, anti-tumor, and anti-inflammatory activities. The enzymatic production of chitooligosaccharides may increase the interest in their potential biomedical or agricultural usability in terms of the safety and simplicity of the manufacturing process. Crab-eating monkey acidic chitinase (CHIA) is an enzyme with robust activity in various environments. Here, we report the efficient degradation of chitin and chitosan by monkey CHIA under acidic and high-temperature conditions. Monkey CHIA hydrolyzed α-chitin at 50 °C, producing N-acetyl-d-glucosamine (GlcNAc) dimers more efficiently than at 37 °C. Moreover, the degradation rate increased with a longer incubation time (up to 72 h) without the inactivation of the enzyme. Five substrates (α-chitin, colloidal chitin, P-chitin, block-type, and random-type chitosan substrates) were exposed to monkey CHIS at pH 2.0 or pH 5.0 at 50 °C. P-chitin and random-type chitosan appeared to be the best sources of GlcNAc dimers and broad-scale chitooligosaccharides, respectively. In addition, the pattern of the products from the block-type chitosan was different between pH conditions (pH 2.0 and pH 5.0). Thus, monkey CHIA can degrade chitin and chitosan efficiently without inactivation under high-temperature or low pH conditions. Our results show that certain chitooligosaccharides are enriched by using different substrates under different conditions. Therefore, the reaction conditions can be adjusted to obtain desired oligomers. Crab-eating monkey CHIA can potentially become an efficient tool in producing chitooligosaccharide sets for agricultural and biomedical purposes.     

壳聚糖制备方法的比较研究_

甲壳素的化学名称为 ( 1 ,4) 2 乙酰胺基 2 脱氧 β Ｄ葡萄糖。当甲壳素通过脱乙酰基反应转变为壳聚糖时 ,由于游离胺基的产生 ,应用性大为增加。壳聚糖分子链上的胺基和羟基都是很好的配位基团 ,使其具有很多纤维素不具有的用途 ,它既是一种天然的高分子螯合剂 ,可与重金属离子如Ｈｇ2 + 、Ｃｕ2 + 、Ａｇ+ 形成稳定的螯合物 ,用于提取回收金属和从污水中去除有害的重金属离子[1,2 ] ,又是一种天然的阳离子型絮凝剂 ,能使水中的悬浮物凝聚而沉降 ,用于污水的净化处理[3] 。表征壳聚糖性能的主要参数有 :脱乙酰度和分子量 ,它们都受甲壳…     

The Effect of Chitin Alkaline Deacetylation at Different Condition on Particle Properties

The abundant biopolymer chitin, found mainly in crustaceous exoskeleton, such as crab, shrimp and lobster, can be deacetylated to yield chitosan. This slightly different biopolymer is more reactive than chitin, being more effective for many applications in fields as environmental remediation, biomedical sciences, catalysis and so on. The main process for chitin deacetylation used sodium hydroxide solutions at high temperatures for long times to obtain chitosan with high deacetylation degree (DD). The present study has evaluated the effect from room temperature (RT), 363 and 393 K, hydroxide concentration (2.0 or 10.0 mol dm³) and time (3 and 24 h) on shrimp chitin deacetylation. Similar amounts of chitin and sodium hydroxide solutions were stirred jointly and the resultant solids were filtered and washed until pH 7, than dried at environmental conditions. The obtained samples were characterized by several techniques, such as elemental analysis, X-rays diffraction (XRD), laser scattering and absorption spectroscopy at infrared region with Fourier transform (FTIR), which was used for DD calculation. The results showed that all chitin-chitosan samples did not reach DD > 90%, as observed for some good commercial chitosans. The highest DD was obtained by the sample prepared at more drastic conditions, as expected, however the higher sodium hydroxide concentration leads to decrease of molecular mass when associated with high temperatures. The crystallinity was influenced mostly by reaction time, which change the positions and intensities as indicated by XRD main peaks, located at 9.3 and 19.4° 2Θ. Particle sizes were strongly diminished by treatment at 393 K, what imply also some increase at the pressure, favoring chain dissociation reactions. This work mapped several properties for chitin-chitosan samples achieved by the described conditions.     

壳聚糖的结构特性及其衍生物的应用

壳聚糖由甲壳素经脱乙酰基而得,又称为可溶性甲壳素。壳聚糖的结构特征使其具有了独特的物理化学性质和生物活性。本文介绍了壳聚糖的结构特性、重要的化学性质及衍生物的应用。     

凝胶渗透色谱法研究壳聚糖生物材料酶降解过程的均匀性

壳聚糖是一种重要的生物医用材料,脱乙酰度是影响其生物降解性能的重要因素。运用凝胶渗透色谱研究了脱乙酰 度及相对分子质量分布相似、而聚合单元N-乙酰氨基-D-葡萄糖和D-氨基葡萄糖分布不同的两种壳聚糖材料在溶菌酶作用 下的降解过程,分析检测了壳聚糖材料在降解过程中的重均相对分子质量、相对分子质量多分散性和相对分子质量分布 的变化。发现聚合单元为随机分布的壳聚糖样品,其降解是均匀的;而聚合单元为段状分布的壳聚糖样品,其降解是非 均匀的;表明其聚合单元的分布方式决定壳聚糖材料酶降解过程的均匀性。     

Squid chitin as a potential alternative chitin source: Deacetylation behavior and characteristic properties

β-Chitin was isolated from squid pens, and the characteristic chemical and physical properties were elucidated in comparison with those of shrimp chitin, α-chitin. Deacetylation behavior of the squid chitin was first studied to look into the reactivity of β-chitin and also to establish an efficient procedure for preparing squid chitosan. The squid chitin proved to show much higher reactivity in alkaline deacetylation than shrimp chitin. Although it was deacetylated quite easily, the product assumed a dark brown color under the ordinary reaction conditions for shrimp chitosan. Squid chitosan was successfully prepared by repeated alkaline treatments under mild conditions, particularly with high concentration alkali at low temperatures, without appreciable discoloration. The structural characteristics of the squid chitin were discussed on the basis of the IR and x-ray analysis data. The crystalline structure of squid chitin was destroyed easily on deacetylation compared to that of shrimp chitin, and moreover, the resulting squid chitosan was amorphous unlike crystalline shrimp chitosan. The squid chitin was characterized by the remarkable affinity for organic solvents and water. Squid chitin and chitosan also showed much higher hygroscopicity and retention of the absorbed water than shrimp chitin and chitosan and are considered to be useful as highly hydrophilic materials. © 1993 John Wiley & Sons, Inc.     

甲壳素/甲壳胺的聚集态结构及性能

制备了不同脱乙酰度的甲壳素,并对脱酰化反应进行了研究,找出了适合不同脱酰度甲壳素的溶剂,探讨了制样温度与甲壳胺膜的结晶形态和力学性能之间的关系,比较了甲壳素、甲壳胺及不同来源甲壳素的结晶形态.     

蘑菇中甲壳素的提取及结构研究

以蘑菇为原料提取甲壳素,并制备壳聚糖。通过滴定法测定由蘑菇制备的壳聚糖的脱乙酰度,用乌氏黏度计测定了比浓黏度,并研究了制备工艺中加热温度和碱处理时间对它们的影响,计算了其产率;对以蘑菇为原料制取的甲壳素、壳聚糖的结构通过红外光谱进行表征。结果表明,在碱处理时间为24h、加热温度为100℃的条件下有较高的脱乙酰度;比浓黏度随着碱处理时间的延长、加热温度的增加都呈下降的趋势;壳聚糖产率为1．69％。制取的甲壳素、壳聚糖的红外光谱图表明,甲壳素在蘑菇中主要是以α-构型存在,α-构型甲壳素在浓碱中经过脱乙酰后生成β-构型的壳聚糖。     

不同脱乙酰度对壳聚糖表面物理及吸附性能的影响

壳聚糖是甲壳素部分脱乙酰化的产物,脱乙酰度的大小对壳聚糖的性能影响很大。利用碱液法,通过对反应时间的控制,制备了不同脱乙酰度的壳聚糖并对其性能进行了研究。结果表明,随着脱乙酰度的增大,壳聚糖膜的吸水率增加,而对其表面接触角的变化则没有影响。同时,壳聚糖海绵对Ca2+和牛血清蛋白(BSA)的吸附能力也随着脱乙酰度的增大而增加。     

Functionalization of chitosan polymer and their applications

The deacetylated derivative of chitin i.e. chitosan is an advantageous and interesting bioactive polymer. Despite its biodegradability, it consists of many reactive primary and secondary hydroxyl (–OH) and amino (–NH₂) functional groups which allow the possibilities of chemical modifications. The several chemical modifications such as alkylation, acylation, quaternization, phthaloylation, sulfation, thiolation, carboxymethylation, graft copolymerization etc. carried out. The chemical modification results various types of derivatives with modified properties for specific applications in varied area mainly of pharmaceutical, biomedical, biotechnological, cosmetic, agricultural, food and non-food industries as well as in water treatment, paper, and textile industry. The ability of chitosan to undergo versatile modifications and applications presents a great opportunity to scientific community and to industry.     

壳聚糖的γ射线辐射降解研究

动力学;无规降解;断链机理;壳聚糖的γ射线辐射降解研究     

LOW MOLECULAR WEIGHT O-CARBOXYMETHYLATED CHITOSANS DERIVED FROM IRRADIATED CHITOSAN AND THEIR ANTIBACTERIAL ACTIVITY

INTRODUCTIONChitin is the second most naturally abundant biopolymer and is found in a variety of organisms, including fungalcell walls, the exoskeleton of crustaceans, skeletal tissue of mollusks and the integument of insects.When treated with alkali, chitin can be deactylated and turned into chitosan, which is a linear binaryheteropolysaccharide composed of (1-4) linked 2-acetamido-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-D-glucopyranose residues. Chitosan has a wide variety of …     

Preparation,Characterization and Optimization of Chitosan Nanoparticles as Carrier for Immobilization of Thermophilic Recombinant Esterase

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres’ average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60°C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).     

水溶性甲壳素及其膜的制备与表征

通过对高脱乙酰度壳聚糖进行均相乙酰化反应，制得脱乙酰度为51．7％的水溶性甲壳素，产物有很好的水溶性．通过红外光谱、X-射线衍射、差热分析等测试表征了水溶性甲壳素结构．结果表明，水溶性甲壳素的结构发生了较大变化，结晶性显著下降是导致水溶性增加的主要原因．     

Cu(Ⅱ)对壳聚糖的配位控制降解

对壳聚糖进行液态均相络合反应制得壳聚糖铜配合物,IR、UV、元素分析及热重分析等检测证实了壳聚糖铜配合物中配位键的存在,且显示壳聚糖在形成配位结构后存在有利于降解的优势构象。以H₂O₂对壳聚糖-Cu(Ⅱ)络合物及壳聚糖进行氧化降解,考察降解过程中粘度的变化及降解产物分子量分布,在相同的降解条件下,壳聚糖铜配合物的降解速度明显高于壳聚糖,降解产物分子量分布较壳聚糖直接降解窄,结果进一步证明壳聚糖铜配合物中存在有利于降解的优势结构,同时证明以金属离子Cu(Ⅱ)对壳聚     

LOW MOLECULAR WEIGHT O-CARBOXYMETHYLATED CHITOSANS DERIVED FROM IRRADIATED CHITOSAN AND THEIR ANTIBACTERIAL ACTIVITY*

 Original chitosan with M_v of 2.7 × 10⁵ was degraded by irradiation with γ-rays and a series of low molecular weight O-carboxymethylated chitosans (O-CMCh) were prepared based on the irradiated chitosan. A kinetic model of the irradiation of chitosan was put forward. Results show that the irradiation degradation of chitosan obeys the rule of random degradation and the degree of deacetylation of irradiated chitosan is slightly raised. The antibacterial activity of O-CMCh is significantly influenced by its MW, and a suppositional antibacterial peak appears when M_v is equal to 2 × 10⁵.     

Preparation and release characteristics of cisplatin albumin microspheres containing chitin and treated with chitosan

Cisplatin (CDDP) containing albumin microspheres and microcapsules incorporating biodegradable macromolecules, chitin and chitosan, were prepared, and their CDDP content and releasing ability and susceptibility to various enzymes were examined. Chitin was incorporated during preparation of the microspheres, while chitosan was used to treat preformed microspheres. CDDP content was remarkably increased by chitin; when chitin was incorporated at a concentration of 1.5%, the CDDP content of the microspheres was found to be 16.2% (1.8 times that with no addition of chitin). CDDP release was suppressed by chitin and chitosan. The 50% CDDP release time was about 1.5 h when no chitin was added, but about 16 h was required when chitin was incorporated into the microspheres at a concentration of 1.5%. Chitin and chitosan suppressed the decomposition by protease. The microspheres treated with 70% deacetylated chitosan showed the greatest susceptibility to lysozyme. In conclusion, CDDP release can be controlled by the use of chitin or chitosan, and the microspheres should show no immunogenicity in vivo because of their susceptibility to lysozyme.     

Preparation and Properties of Alginate/Water‐Soluble Chitin Blend Fibers

Water‐soluble chitin (half‐deacetylated chitin) was prepared from chitosan by N‐acetylation with acetic anhydride. Alginate/water‐soluble chitin blend fibers were prepared by spinning their mixture solution through a viscose‐type spinneret into a coagulating bath containing aqueous CaCl₂ and ethanol. The structure and properties of the blend fibers were studied with the aids of infrared spectra (IR), X‐ray diffraction (XRD) and scanning electron microscopy (SEM). structure analysis indicated good miscibility existed between alginate and water‐soluble chitin, due to the strong interaction from the intermolecular hydrogen bonds and electrostatic interactions. Best values for the dry tensile strength and breaking elongation were obtained when the water‐soluble chitin content was 30 wt%. The wet tensile strength and breaking elongation decreased with the increase of water‐soluble chitin content. The introduction of water‐soluble chitin in the blend fiber can improve the water‐retention properties of the blend fiber compared to pure alginate fiber. The fibers treated with aqueous solution of silver nitrate have good antibacterial activity to Staphylococcus aureus.     

Effects of Chitin/Chitosan and Their Oligomers/Monomers on Migrations of Macrophages

The effects of chitin/chitosan and their oligomers/monomers on the migrations of mouse peritoneal macrophage (PEM) and the rat macrophage cell line (rMp) were evaluated in vitro. In direct migratory assay using the blind well chamber method, the migratory activity of PEM was enhanced significantly by chitin and chitosan oligomers (NACOS and COS, respectively), but reduced by chitin, chitosan, and the chitosan monomer (GlcN). The migratory activity of rMp was increased significantly by chitin, chitosan, and the polymers, NACOS and GlcN.