Effects of degree of deacetylation on enzyme immobilization in hydrophobically modified chitosan

Chitosan is a deacetylated form of the polysaccharide chitin. Over the last decade, researchers have employed reductive amination to hydrophobically modify chitosan to induce a micellar structure. These micellar polymers have been used for a variety of purposes including drug delivery and enzyme immobilization and stabilization. However, commercial sources of chitosan vary in their degree of deacetylation and there remains a paucity of information regarding how this can impact the modified polymer’s functionality for enzyme immobilization. This paper, therefore, evaluates the effect that the degree of deacetylation has on the hydrophobic modification of medium molecular weight chitosan via reductive amination with long chain aldehydes and the resulting changes in enzyme activity after the immobilization of glucose oxidase in the micellar polymeric structure. The chitosan was deacetylated to differing degrees via autoclaving in 40–45% NaOH solutions and characterized using NMR, viscosity measurements, and differential scan calorimetry. Results suggest that a high degree of deacetylation provides optimal enzyme immobilization properties (i.e. high activity), but that the deacetylation method begins to significantly decrease the polymer molecular weight after a 20 min autoclave treatment, which negatively affects immobilized enzyme activity.     

Microwave technique for efficient deacetylation of chitin nanowhiskers to a chitosan nanoscaffold

A chitosan nanoscaffold in the form of a colloidal solution was obtained from the deacetylation of chitin whiskers under alkaline conditions by using a microwave technique in only 1/7 of the treatment time of the conventional method. Fourier-transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (¹H NMR) techniques confirm the degree of deacetylation to be above 90% within 3 h. The wide-angle X-ray diffraction (WAXD) pattern clearly shows that the highly crystalline chitin whiskers are changed to amorphous chitosan. SEM micrographs show the aggregation of branched nanofibers, whereas the TEM micrographs reveal the scaffold morphology.     

Characteristics of deacetylation and depolymerization of β-chitin from jumbo squid (Dosidicus gigas) pens

This study evaluated the deacetylation characteristics of β-chitin from jumbo squid (Dosidicus gigas) pens by using strongly alkaline solutions of NaOH or KOH. Taguchi design was employed to investigate the effect of reagent concentration, temperature, time, and treatment step on molecular mass (MM) and degree of deacetylation (DDA) of the chitosan obtained. The optimal treatment conditions for achieving high MM and DDA of chitosan were identified as: 40% NaOH at 90 °C for 6 h with three separate steps (2 h + 2 h + 2 h) or 50% NaOH at 90 °C for 6 h with one step, or 50% KOH at 90 °C for 4 h with three steps (1 h + 1 h + 2 h) or 6 h with one step. The most important factor affecting DDA and MM was temperature and time, respectively. The chitosan obtained was then further depolymerized by cellulase or lysozyme with cellulase giving a higher degradation ratio, lower relative viscosity, and a larger amount of reducing-end formations than that of lysozyme due to its higher susceptibility. This study demonstrated that jumbo squid pens are a good source of materials to produce β-chitosan with high DDA and a wide range of MM for various potential applications.     

Shrimp chitin as substrate for fungal chitin deacetylase

The fungal chitin deacetylases (CDA) studied so far are able to perform heterogeneous enzymatic deacetylation on their solid substrate, but only to a limited extent. Kinetic data show that about 5-10% of the N-acetyl glucosamine residues are deacetylated rapidly. Thereafter enzymatic deacetylation is slow. In this study, chitin was exposed to various physical and chemical conditions such as heating, sonicating, grinding, derivatization and interaction with saccharides and presented as a substrate to the CDA of the fungus Absidia coerulea. None of these treatments of the substrate resulted in a more efficient enzymatic deacetylation. Dissolution of chitin in specific solvents followed by fast precipitation by changing the composition of the solvent was not successful either in making microparticles that would be more accessible to the enzyme. However, by treating chitin in this way, a decrystallized chitin with a very small particle size called superfine (SF) chitin could be obtained. This SF chitin, pretreated with 18% formic acid, appeared to be a good substrate for fungal deacetylase. This was confirmed both by enzyme-dependent deacetylation measured by acetate production as well as by isolation and assay for the degree of deacetylation (DD). In this way chitin (10% DD) was deacetylated by the enzyme into chitosan with DD of 90%. The formic acid treatment reduced the molecular weight of the polymeric chain from 2x10(5) in chitin to 1.2 x 10(4) in the chitosan product. It is concluded that nearly complete enzymatic deacetylation has been demonstrated for low-molecular chitin.     

Preparation of low-molecular-weight chitosan using phosphoric acid

Two types of low degree of polymerisation (DP) chitosan were prepared by homogeneous hydrolysis of chitosan in 85% phosphoric acid at room temperature for 1–6 weeks. The hydrolysates were collected by addition of excess ethanol, and were fractionated by solubility in water. The changes in yields of water-insoluble (higher DP) and water-soluble (lower DP) fractions were determined as a function of hydrolysis time. The hydrolysis proceeded with further deacetylation of chitosan, resulting in degree of deacetylation of more than 90%. The water-insoluble fraction prepared after the hydrolysis for 4 weeks (43% yield) had a weight-average DP ( ) of 16·8, and showed the ‘tendon’ type X-ray diffraction pattern. The water-soluble fraction (12·5% yield) had a of 7·3, and showed the ‘annealed’ type pattern.     

The antitumor activity of the hydrolysates of chitinous materials hydrolyzed by crude enzyme from Bacillus amyloliquefaciens V656

Chitin, colloidal chitin and water-soluble chitosan were hydrolyzed by crude enzyme solution produce by Bacillus amyloliquefaciens V656. The hydrolysates with 12 h hydrolysis contained optimal (GlcNAc)₆ and showed higher antitumor activity. Among those chitinous materials, the most effective one was the hydrolysates of water-soluble chitosan, which inhibited the growth of CT26 cells and reduced the survival rate to 34% in 1 day. Since the hydrolysate of water-soluble chitosan contained the optimal hexamer/(GlcNAc)₆ at 12 h, it is conjectured that the antitumor activity should be related to (GlcNAc)₆. This conjecture was further affirmed by experiment with pure (GlcNAc)₆. However, This phenomenon might be due to the synergistic effect of the oligomers (GlcNAc)_n, n = 1–6 in the hydrolysates. The antitumor effect of the chitinous hydrolysates is worth further investigation.The aim of this study was to investigate the induced apoptosis in CT26 cells by the hydrolysates of chitinous materials. It was found that the hydrolysates (A, B and C) inhibited the survival of CT26 cells in a concentration- and time-dependent manner. The hydrolysates induced characteristic DNA fragmentation of the CT26 cells. These results suggested that the hydrolysates from chitinous materials are potent apoptosis-inducing agents for CT26 cells.     

Antimicrobial textile treated with chitosan from Aspergillus niger mycelial waste

The waste biomass of Aspergillus niger, following citric acid production, was used as a source for fungal chitosan extraction. The produced chitosan was characterized with deacetylation degree of 89.6%, a molecular weight of 25,000 dalton, 97% solubility in 1% acetic acid solution and comparable FT-IR spectra to standard shrimp chitosan. Fungal chitosan was applied as a cotton fabric finishing agent using pad-dry-cure method. The topographical structure of chitosan-treated fabrics (CTF) was much improved compared with control fabrics. CTF, after durability tests, exhibited a powerful antimicrobial activity against both E. coli and Candida albicans, the captured micrographs for E. coli cells contacted with CTF showed a complete lysis of cell walls with the prolonging contact time. The produced antimicrobial CTF could be proposed as a suitable material for many medical and hygienic applications.     

Chitosan topical gel formulation in the management of burn wounds

Wound healing properties of chitosan with different molecular weight and degree of deacetylation ranges have been examined. The macroscopic image and histopathology were examined using chitosan, Fucidin^® ointment and to blank. The rate of contraction was evaluated by determination of the unclosed area as a function of time. The treated wounds were found to contract at the highest rate with high molecular weight–high degree of deacetylation chitosan-treated rats as compared to untreated, treated, and Fucidin^® ointment-treated rats. Wounds treated with high molecular weight chitosan had significantly more epithelial tissue (p < 0.05) than wounds with any other treatment and the best re-epithelization and fastest wounds closure were found with the high molecular weight chitosan treatment group. Histological examination and collagenase activity studies revealed advanced granulation tissue formation and epithelialization in wounds treated with high molecular weight chitosan (p < 0.05). High molecular weight with high degree of deacetylation chitosan samples therefore demonstrates potential for use as a treatment system for dermal burns.     

Effect of ultrasonic treatment on the biochemphysical properties of chitosan

Four chitosans with different molecular weights and degrees of deacetylation degree and 28 chitosans derived from these initial chitosans by ultrasonic degradation have been characterized by gel permeation chromatography (GPC), FT-IR spectroscopy, X-ray diffraction and titrimetric analyses. Antimicrobial activities were investigated against E. coli and S. aureus using an inhibitory rate technique. The results showed that ultrasonic treatment decreased the molecular weight of chitosan, and that chitosan with higher molecular weight and higher DD was more easily degraded. The polydispersity decreased with ultrasonic treatment time, which was in linear relationship with the decrease of molecular weight. Ultrasonic degradation changed the DD of initial chitosan with a lower DD (<90%), but not the DD of the initials chitosan with a higher DD (>90%). The increased crystallinity of ultrasonically treated chitosan indicated that ultrasonic treatment changed the physical structure of chitosan, mainly due to the decrease of molecular weight. Ultrasonic treatment enhanced the antimicrobial activity of chitosan, mainly due to the decrease of molecular weight.     

Chemical profiling of the deacetylase activity of acetyl xylan esterase A (AxeA) variants on chitooligosaccharides using hydrophilic interaction chromatography-mass spectrometry

Chitosan oligosaccharides (oligomers of (GlcNAc)_x(GlcN)_y) are used in the pharmaceutical, cosmetic and food industries and are reported to have therapeutic benefits. However, it is unknown whether their biological activity depends on the degree of deacetylation or the sequence of residues within the oligomer. We report here the development of a random mutagenesis method for directed evolution of Streptomyces lividans acetyl xylan esterase (AxeA), which we previously showed is able to deacetylate chitinous substrate, in order to obtain chitooligosaccharides with well-defined structural properties. A colorimetric assay was used to pre-screen libraries for p-nitrophenol acetate hydrolysis activity and an HPLC-UV absorbance assay was optimized to subsequently screen for deacetylase activity toward hexa-N-acetyl-glucosamine substrate (GlcNAc)₆. Native AxeA and two variants displaying > 50% deacetylation of the oligohexamer substrate after reaction at 50 °C for 24 h in diluted culture supernatant were then selected for detailed analysis of the enzymatic products. A HILIC (hydrophilic interaction chromatography)-mode LC method was developed for profiling the deacetylated chitooligosaccharide products and HILIC-MS/MS sequencing revealed that ca. 30 different deacetylation products ranging from (GlcNAc)₅(GlcN)₁ to (GlcNAc)₁(GlcN)₅ and isomers thereof were produced. The AxeA variants produced, on average, 26% more unique products than the native enzyme; however, none were able to fully deacetylate the substrate to make (GlcN)₆. The long term goal of this multidisciplinary approach is to improve the activity of chitosan oligosaccharides to an industrially applicable level.     

Extraction and characterization of chitin and chitosan from local sources

Chitin has been extracted from six different local sources in Egypt. The obtained chitin was converted into the more useful soluble chitosan by steeping into solutions of NaOH of various concentrations and for extended periods of time, then the alkali chitin was heated in an autoclave which dramatically reduced the time of deacetylation. Chitin from squid pens did not require steeping in sodium hydroxide solution and showed much higher reactivity towards deacetylation in the autoclave that even after 15 min of heating a degree of deacetylation of 90% was achieved. The obtained chitin and chitosan were characterized by spectral analysis, X-ray diffraction and thermo gravimetric analysis.     

Reactive oxygen species scavenging activity of aminoderivatized chitosan with different degree of deacetylation

Chitosans with different degree of deacetylation were prepared from crab shell chitin in the presence of alkali. Aminoderivatized chitosan derivatives were prepared in addition of amino functional groups at a hydroxyl site in the chitosan backbone. Six kinds of aminoderivatized chitosan such as aminoethyl-chitosan (AEC90), dimethylaminoethyl-chitosan (DMAEC90), and diethylaminoethyl-chitosan (DEAEC90), which were prepared from 90% deacetylated chitosan, and AEC50, DMAEC50 and DEAEC50, which were prepared from 50% deacetylated chitosan, were prepared and their reactive oxygen species (ROS) scavenging activities were investigated against hydroxyl radical, superoxide anion radical and hydrogen peroxide. The electron spin resonance (ESR) spectrum revealed that AEC90 showed the highest scavenging effects against hydroxyl and superoxide anion radical, the effects were 91.67% and 65.34% at 0.25 and 5 mg/mL, respectively. For hydrogen peroxide scavenging effect, DEAEC90 exhibited the strongest activity. These results suggest that the scavenging effect depends on their degree of deacetylation and substituted group.     

Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry

The degree of acetylation of chitosan can be determined in acetic acid solutions (～0·01m) containing 1 g dry chitosan per litre by first derivative ultraviolet spectrophotometry at 199 nm. At this wavelength, the N-acetylglucosamine absorbance readings are linearly dependent on concentration and are not influenced by the presence of acetic acid. Correction factors for the contribution of glucosamine in highly deacetylated chitosans can be easily derived. Typical results for the chitosan of Euphausia superba are: degree of acetylation, 42·6; relative standard deviation, 1·3%; confidence limits, ±0·7. This method is simpler, more precise and faster than the infrared method. Sonication of chitosan solutions leads to immediate chain degradation and to detectable deacetylation after more prolonged periods of time, especially when the pH is 1·0.     

Chitin/chitosan transformation by thermo-mechano-chemical treatment including characterization by enzymatic depolymerization

Chitosan, the deacetylated derivative of chitin, was until recently produced by hydrolysis in 50% (w/v) NaOH. Application of thermo-mechano-chemical technology to chitin deacetylation was evaluated as an alternative method of chitosan production. This process consists of a cascade reactor unit operating under reduced alkaline conditions of 10% (w/v) NaOH. Prior mercerization of chitin at 4 degrees C for 24 h was required for high deacetylation yields. Sudden decompression of the aqueous alkaline suspension of mercerized chitin resulted in near complete deacetylation of chitin. Reactor residence time was 90 s at 230 degrees C prior to decompression. The chitosan produced was characterized by elemental analysis, (13)C-NMR and enzymatic depolymerization. Enzymatic determination of the degree of acetylation of chitin/chitosan mixtures was also investigated. Relative chitinase and/or chitosanase digestibilities were shown to be strongly dependent on chitin deacetylation. Based on enzymatic digestibilities, the alkaline aqueous high shear process does not appear to produce significant secondary products. Correlation of chitosanase digestibility with percentage of deacetylation provides a simple biological assay to study chitosan composition.     

胍乙酸壳聚糖的合成及其对黄瓜的保鲜研究

以自制的不同脱乙酰度的壳聚糖和1-氯胍乙酸为原料合成了胍乙酸壳聚糖,研究了胍乙酸壳聚糖对黄瓜的保鲜效果。结果表明,由脱乙酰度为96%的壳聚糖制得的胍乙酸壳聚糖平均分子量为5287。随着脱乙酰度的增加,黄瓜失重率的增加逐渐减缓,随着贮存时间延长总叶绿素含量先升高然后缓慢下降,而维生素C含量则一直缓慢下降;脱乙酰度为96%的壳聚糖制得的胍乙酸壳聚糖贮存35 d后,黄瓜的质量损失为0.7%;贮存20 d后,总叶绿素含量仍然可达1.34 mg/g;贮存时间20 d后,维生素C含量可达0.18 mg/g。     

半干法微波处理制备壳聚糖

本文报导了采用微波处理半干法由甲壳素脱乙酰制备壳聚糖。与传统方法相比,反应时间大大缩短,降低了生产成本。并讨论了影响壳聚糖脱乙酰度和粘度的因素,例如,反应时间,碱浓度,料比。在较适宜的条件下,7分钟内脱乙酰度可大于75％。     

Production and characterization of chitosan obtained from <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> grown on potato chip processing waste

Potato chip processing waste of trimmed potato, potato peel and substandard (low-quality) potato chips, obtained from a potato chip processing plant, were used as substrates for chitosan production from Rhizopus oryzae. It was cultured on each waste product at 30 ± 2°C and 70% moisture content for 21 days. Fermented potato peel had the highest yield after 5 days of fermentation. The cultivation condition of chitosan obtained from R. oryzae was optimum for a peel size of less than 6 mesh, 70% moisture content and a pH of 5. Furthermore, the best extraction condition was using 46% sodium hydroxide at 46°C for 13 h followed by 2% acetic acid at 95°C for 8 h. The maximum chitosan yield obtained by these conditions was 10.8 g/kg substrate. Fungal chitosan properties were found to be 86–90% degree of deacetylation, molecular weight of 80–128 kDa and viscosity of 3.1–6.1 mPa s. Therefore, potato peel could be applied as a low cost substrate for chitosan production from R. oryzae.     

High yield production of monomer-free chitosan oligosaccharides by pepsin catalyzed hydrolysis of a high deacetylation degree chitosan

The high molecular weight of chitosan, which results in a poor solubility at neutral pH values and high viscosity aqueous solutions, limits its potential uses in the fields of food, health and agriculture. However, most of these limitations are overcome by chitosan oligosaccharides obtained by enzymatic hydrolysis of the polymer. Several commercial enzymes with different original specificities were assayed for their ability to hydrolyze a 93% deacetylation degree chitosan and compared with a chitosanase. According to the patterns of viscosity decrease and reducing end formation, three enzymes--cellulase, pepsin and lipase A--were found to be particularly suitable for hydrolyzing chitosan at a level comparable to that achieved by chitosanase. Unlike the appreciable levels of both 2-amino-2-deoxy-D-glucose and 2-acetamido-2-deoxy-D-glucose monomers released from chitosan by the other enzymes after a 20h-hydrolysis (4.6-9.1% of the total product weight), no monomer could be detected following pepsin cleavage. As a result, pepsin produced a higher yield of chitosan oligosaccharides than the other enzymes: 52% versus as much as 46%, respectively. Low molecular weight chitosans accounted for the remaining 48% of hydrolysis products. The calculated average polymerization degree of the products released by pepsin was around 16 units after 20h of hydrolysis. This product pattern and yield are proposed to be related to the bond cleavage specificity of pepsin and the high deacetylation degree of chitosan used as substrate. The optimal reaction conditions for hydrolysis of chitosan by pepsin were 40 degrees C and pH 4.5, and an enzyme/substrate ratio of 1:100 (w/w) for reactions longer than 1h.     

Proteinase inhibitor synthesis in tomato leaves : induction by chitosan oligomers and chemically modified chitosan and chitin

Soluble chemical derivatives of chitin and chitosan including ethylene glycol chitin, nitrous acid-modified chitosan, glycol chitosan, and chitosan oligomers, produced from chitosan by limited hydrolysis with HCl, were found to possess proteinase inhibitor inducing activities when supplied to young excised tomato (Lycopersicon esculentum var Bonnie Best) plants. Nitrous acid-modified chitosans and ethylene glycol chitin exhibited about 2 to 3 times the activity of acid hydrolyzed chitosan and 15 times more activity than glycol chitosan. The parent chitin and chitosans are insoluble in water or neutral buffers and cannot be assayed. Glucosamine and its oligomers from degree of polymerization = 2 through degree of polymerization = 6 were purified from acid-fragmented chitosan and assayed. The monomer was inactive and dimer and trimer exhibited weak activities. Tetramer possessed higher activity and the larger pentamer and hexamer oligomers were nearly as active as the total hydrolyzed mixture. None of the fragments exhibited more than 2% acetylation (the limits of detection). The contents of the acid-fragmented mixture of oligomers was chemically N-acetylated to levels of 13% and 20% and assayed. The N-acetylation neither inhibited nor enhanced the proteinase inhibitor inducing activity of the mixture. These results, along with recent findings by others that chitinases and chitosanases are present in plants, provide further evidence for a possible role of soluble chitosan fragments as signals to activate plant defense responses.     

Purification and characterization of an extracellular chitosanase produced by Amycolatopsis sp. CsO-2

Extracellular chitosanase produced by Amycolatopsis sp. CsO-2 was purified to homogeneity by precipitation with ammonium sulfate followed by cation exchange chromatography. The molecular weight of the chitosanase was estimated to be about 27,000 using SDS-polyacrylamide gel electrophoresis and gel filtration. The maximum velocity of chitosan degradation by the enzyme was attained at 55°C when the pH was maintained at 5.3. The enzyme was stable over a temperature range of 0–50°C and a pH range of 4.5–6.0. About 50% of the initial activity remained after heating at 100°C for 10 min, indicating a thermostable nature of the enzyme. The isoelectric point of the enzyme was about 8.8. The enzyme degraded chitosan with a range of deacetylation degree from 70% to 100%, but not chitin or CM-cellulose. The most susceptible substrate was 100% deacetylated chitosan. The enzyme degraded glucosamine tetramer to dimer, and pentamer to dimer and trimer, but did not hydrolyze glucosamine dimer and trimer.