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The effect of chitosan decoration on the transport of epigallocatechin (EGC)-encapsulated ferritin cage across the Caco-2 cells was investigated. After the encapsulation of EGC in apo-red bean (adzuki) ferritin (apoRBF), the EGC-loaded apoRBF nanoparticle (ER) was fabricated with an encapsulation ratio of 11.6% (w/w). The results indicated that different chitosan molecules (with molecular weights of 980, 4600, 46 000, and 210 000 Da) could attach onto the apoRBF via electrostatic interactions to form ER-chitosan complexes (ERCs) (ERCs980, ERCs4600, ERCs46000, and ERCs210000). ERCs980 and ERCs4600 retained the typical shell-like morphology of ferritin with a size distribution of 12 nm and showed weak negative zeta-potentials at pH 6.7, while ERCs46000 and ERCs210000 significantly aggregated. Furthermore, the transport of EGC in ERCs980 and ERCs4600 across the Caco-2 cells was enhanced by the transferrin receptor 1 (TfR-1)-mediated absorption pathway, demonstrating that chitosan molecules with low molecular weights of 980 and 4600 Da were beneficial to the absorption of EGC based on the ferritin cage. This study will facilitate the application of ferritin-chitosan materials for fabricating the core-shell platform for encapsulation and bioavailability enhancement of bioactive molecules.

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3-D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3-D1 was similar to BmCPAP3-A1 and BmCPAP3-C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin-binding protein, BmCPAP3-D1, which exhibits high binding affinity to deacetylated chitin.

Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity.

To know the mechanism of ammonia assimilation in non-heading Chinese cabbage (Brassica campestrish L. ssp. chinensis (L.) Makino) leaves regulated by chitosan (CTS), a CTS-binding protein was isolated from non-heading Chinese cabbage leaves using the chitosan affinity chromatography approach and this CTS-binding protein was partially characterized. The profile of the 53.1 kDa purified protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was compared with the native molecular weight of 106.5 kDa, which indicated that the purified protein was a dimer with identical subunits. After isoelectric focusing, a band was obtained at pH 8.25. The agglutination test and periodic acid-Schiff staining further revealed that the protein was a glycoprotein with lectin activity. Moreover, the purified protein contained 17.4% (w/w) neutral carbohydrate and 82.56% (w/w) protein. The comparison of this protein and the 67 kDa CTS-binding protein isolated previously from Rubus culture tissue exhibited some differences in characterization. According to results of peptide mass fingerprinting analysis, the protein purified in the present study does not show any similarity with any protein in the protein data bank. Thus, it was deduced that the protein purified in the present study is a novel CTS-binding protein. (Managing editor: Li-Hui ZHAO)

Chitin deacetylation is required to make the cuticle rigid and compact through chitin chain crosslinking. Thus it is presumed that specialized proteins are required to bind deacetylated chitin chains together. However, deacetylated-chitin binding proteins have not ever been reported. In a previous work, six cuticular proteins analogous to peritrophin 3 (CPAP3s) were found to be abundant in the moulting fluid of Bombyx mori. In this study, these BmCPAP3s (BmCPAP3-A1, BmCPAP3-A2, BmCPAP3-B, BmCPAP3-C, BmCPAP3-D1 and BmCPAP3-D2) were cloned and expressed in Escherichia coli and purified using metal-chelating affinity chromatography. Their binding activities demonstrated that although all of the BmCPAP3s showed similar binding abilities toward crystalline chitin and colloidal chitin, they differed in their affinities toward partially and fully deacetylated chitin. Amongst them, BmCPAP3-D1 exhibited the highest binding activity toward deacetylated chitin. The gene expression pattern of BmCPAP3-D1 was similar to BmCPAP3-A1 and BmCPAP3-C at most stages except that it was dramatically upregulated at the beginning of the pupa to adult transition stage. This work is the first report of a chitin-binding protein, BmCPAP3-D1, which exhibits high binding affinity to deacetylated chitin.

The functionality of halloysite (Hal) nanotubes as drug carriers can be improved by lumen enlargement and polymer modification. This study investigates the influence of selective acid etching on Hal functionalization with cationic biopolymer chitosan. Hal was subjected to lumen etching under mild conditions, loaded under vacuum with nonsteroidal antiinflammatory drug aceclofenac, and incubated in an acidic solution of chitosan. The functionality of pristine and etched Hal before and upon polymer functionalization was assessed by ζ-potential measurements, structural characterization (FT-IR, DSC and XRPD analysis), cell viability assay, drug loading and drug release studies. Acid etching increased specific surface area, pore volume and pore size of Hal, decreased ζ-potential and facilitated binding of the cationic polymer. XRPD and DSC analysis revealed crystalline structure of etched Hal. Successful chitosan binding and drug entrapment were further confirmed by FT-IR and DSC studies. XRPD showed surface polymer binding. DSC and FT-IR analyses confirmed the presence of the entrapped drug in its crystalline form. Drug loading was increased for ≈81% by selective lumen etching. Slight decrease of drug content occurred during chitosan functionalization due to aceclofenac diffusion in the polymer solution. The drug release was more sustained from etched Hal nanocomposites (up to ≈87% for 12 h) than from pristine Hal (up to ≈97% for 12 h) due to more intensive chitosan binding. High human fibroblast survival rates upon exposure to pristine and etched Hal before and after chitosan functionalization (>90% in the concentration of 1000 μg/mL) confirmed that both lumen etching under mild conditions and polymer functionalization had no significant effect on cytocompatibility. Based on these findings, selective lumen etching in combination with polycation modification appears to be a promising approach for improvement of Hal nanotubes functionality by increasing payload, polymer binding capacity, and sustained release properties with no significant effect on their cytocompatibility.

This work develops a simple and innovative test-strip to monitor antibiotics in aquaculture facilities by an equipment-free approach. It consists of a low-cost disposable cellulose paper that was chemically modified to produce a colour change when in contact with a given antibiotic. In brief, the cellulose substrate was subject to oxidation with periodate, followed by amination with chitosan binding and modification with Cu(II). The test strip was then dipped in the target solution and the intensity of the colour generated therein revealed the concentration of antibiotic present for concentrations higher than 0.5 mM. The higher the concentration in sulfadiazine (SDZ), the more intense the pink colour formed in the final solution, which was also turbid due to the insolubility of the formed product. This colour intensity also varied linearly with the logarithm of the SDZ concentration (from 0.5 to 5 mM), when plotted against the sum of the RGB coordinates extracted from digital pictures. The linear equation of this response was represented by (R+G+B)=256.1 log(SDZ, mol/L)362.0, with an R-squared of 0.9913. The test-strip was stable for at least 15 days and was selective in the presence of tetracycline and difloxacin, while the response to other members of the sulfadiazine family requires prior evaluation. Overall, the test-strips developed herein are inexpensive and provide valuable (semi-) quantitative data for monitoring SDZ in waters, a most valuable approach to control and reduce the level of antibiotics in fish tanks, which in turn may reduce the costs of fish production and the environmental concerns linked to this practice. Moreover, the test strip uses a cellulose substrate that has little environmental impact upon discard., The authors acknowledge funding from project PTDC/AAGTEC/5400/2014, POCI-01-0145-FEDER-016637 funded by European through FEDER (European Funding or Regional Development) via COMPETE2020—POCI (operational program for internationalization and competitively) and by national funding through the National Foundation for Science and Technology, I.P., info:eu-repo/semantics/publishedVersion

The interaction of endotoxin (lipopolysaccharide - LPS) with low molecular weight chitosan (5.5 kDa), its N-acylated derivative and chitoliposomes was studied using a gravimetric piezoelectric quartz crystal microbalance biosensor. The optimal conditions for the formation of a biolayer based on immobilized LPS on the resonator surface and its regeneration were elaborated. The association and dissociation rate constants for LPS binding to chitosans were determined and the affinity constants (Kaf) were calculated based on the data on changes in the oscillation frequency of the quartz crystal resonator. The Kaf values correlated with the ones obtained using other methods. The affinity of N-acylated chitosan binding to LPS was higher than that of the parent chitosan binding to LPS. Based on the results obtained, we suggest that water-soluble N-acylated derivatives of chitosan with low degree of substitution of amino groups could be useful compounds for endotoxin binding and neutralization.

Conjugations of DNA with chitosans 15 kD (ch-15), 100 kD (ch-100) and 200 kD (ch-200) were investigated in aqueous solution at pH 5.5-6.5. Multiple spectroscopic methods and atomic force microscopy (AFM) were used to locate the chitosan binding sites and the effect of polymer conjugation on DNA compaction and particle formation. Structural analysis showed that chitosan-DNA conjugation is mainly via electrostatic interactions through polymer cationic charged NH2 and negatively charged backbone phosphate groups. As polymer size increases major DNA compaction and particle formation occurs. At high chitosan concentration major DNA structural changes observed indicating a partial B to A-DNA conformational transition.

The application of bioactive lutein in the food industry is limited because of its poor water-solubility, instability, and low bioavailability. Nano-sized ferritin and chitosan provide a platform for fabricating shell–core system to encapsulate lutein. Herein, soybean seed ferritin (Glycine max) and chitosan stabilized lutein composites (FCLs) were fabricated by a unique reversible disassembly-reassembly character of apoferritin and ferritin–chitosan interaction. Results showed that lutein molecules were successfully encapsulated within the apoferritin with a molar ratio of 25.2 to 1 (lutein/ferritin), and the encapsulation efficiency and loading capacity were 16.8% and 2.50% (w/w), respectively. It was calculated that approximately 10 chitosan molecules were bound to a ferritin with a binding constant of 6.3 × 105 M−1, suggesting electrostatic interaction played an important role in ferritin–chitosan interaction. Results also indicated as much as 74.1% (w/w) of lutein was retained in FCLs after storage at 20 °C for 7 days. In addition, the photo- and heat-stability of lutein in FCLs were greatly improved as compared to free lutein. Furthermore, FCLs exhibited prolonged release of lutein in simulated gastrointestinal tract (GI) digestion as a result of ferritin coating and chitosan binding. Interestingly, food components exerted different effects in lutein release, EGCG, grape seed proanthocyanidin, and milk could inhibit while pectin could facilitate the release of lutein from FCLs. This work demonstrates an innovative strategy to solubilize, stabilize and control release of functional food nutrients.

A novel β-NaCaPO 4 containing borate glass-ceramic is prepared. Two porous glass-ceramic scaffolds are prepared by binding particles with the size of 200–300 μm by 5 wt.% sodium silicate solution and 2 wt.% chitosan solutions, respectively. The reaction of the scaffolds in the SBF solution is characterized by weight loss analysis, XRD, FTIR, and SEM. The same is done to the 45S5 glass scaffolds as comparison. XRD and FTIR indicate that the carbonate hydroxyapatite has formed more rapidly on the borate glass-ceramic scaffolds. The carbonated hydroxyapatite depositing on chitosan binding scaffolds has lower crystallization degree than that on sodium silicate binding scaffolds and is similar to that of the human bone, which makes the chitosan binding scaffolds a good potential prospect in the field of tissue engineering.

To reduce costs and improve practicability, an ion imprinted magnetic chitosan (IMCS) was synthesized through co-precipitation using steel pickling waste liquor and chitosan and Cu(II) as template ions, which was then characterized by TEM, SEM, EDX, FTIR, XRD and VSM. The batch experiments were carried out for its potential application and high selectivity of Cu(II) removal, which were observed due to the paramagnetic properties and coordination reactions in the imprinted cavities. Kinetic studies revealed that the adsorption process followed a pseudo second-order model and the equilibrium data fit perfectly with the Langmuir isotherm model. The maximum adsorption capacity was 109.89 mg g−1. Negative values for ΔH0 and ΔG0 indicated an exothermic and spontaneous adsorption process. The adsorption process was found to be a chemical reaction and coordination complexes were formed between the metal ions and the groups of chitosan binding mainly in the “bridge model”. Moreover, 0.2 mol L−1 HCl solution was considered as the most appropriate eluent for regeneration. It showed a great performance in the experiments for practical copper wastewater and the process was considerably cost-effective.

The main objective of this study was to obtain chitosan functionalized viscose fabric with improved antibacterial properties and washing durability. In this regard carboxyl and aldehyde groups, as binding points for irreversible chitosan attachment into/onto viscose fabric, were introduced by two different pretreatments: 2,2,6,6-tetramethylpiperidine-1-oxy radical (TEMPO) oxidation and coating with TEMPO oxidized cellulose nanofibrils (TOCN). The Fourier transform infrared spectroscopy, elemental analysis, zeta potential measurements, scanning electron microscopy, breaking strength and antibacterial testing were used to evaluate the influence of these pretreatments on chitosan binding, but also on chemical, electrokinetic, morphological, mechanical and antibacterial properties of pretreated and chitosan functionalized viscose fabrics. Washing durability of chitosan functionalized viscose was monitored through changes in the chitosan content, electrokinetic and antibacterial properties after multiple washing. TOCN coating improves mechanical properties of fabric, while TEMPO oxidation deteriorates them. The results show that both pretreatments improve chitosan adsorption and thus antibacterial properties, which are highly durable to washing. After five washings, the chitosan functionalized pretreated viscose fabrics preserve their antibacterial activity against Staphylococcus aureus, while antibacterial activity against Escherichia coli was lost. TOCN coated and chitosan functionalized viscose fabric is a high value-added product with simultaneously improved antibacterial and mechanical properties, which may find application as medical textiles.

Oxygen and carbon dioxide plasma treatments were applied in order to study their influences on chitosan adhesion onto PET foils. Modification of the surface and the adsorption of chitosan were monitored by the determination of XPS spectra, titrations, zeta potential, contact angles, and AFM. The plasma treatment resulted in hydrophilization of the surface through the introduction of new polar oxygen-containing groups. The treatment also yielded binding-sites for amino groups of chitosan. This led to a decrease in accessible protonated amino groups after chitosan binding onto plasma-activated foils in comparison with the plasma-non-activated ones. Consequently, the layer of chitosan adsorbed after plasma treatment was less antimicrobially active in the case of O2-treated foils. Surprisingly, the layer of chitosan adsorbed after CO2 plasma treatments were, in general, more active as antimicrobial agents than those of the non-plasma treated PET surfaces.

Background and objectives A current public health issue is preventing post-surgical complications by designing antibacterial implants. To achieve this goal, in this study we evaluated the antibacterial activity of an animal-free chitosan grafted onto a titanium alloy. Methods Animal-free chitosan binding on the substrate was performed by covalent link via a two-step process using TriEthoxySilylPropyl Succinic Anhydride (TESPSA) as the coupling agent. All grafting steps were studied and validated by means of X-ray Photoelectron Spectroscopy (XPS), Time-of-Flight secondary ion mass spectrometry (ToF-SIMS) analyses and Dynamic-mode Secondary Ion Mass Spectrometry (DSIMS). The antibacterial activity against Escherichia coli and Staphylococcus aureus strains of the developed coating was assessed using the number of colony forming units (CFU). Results XPS showed a significant increase in the C and N atomic percentages assigned to the presence of chitosan. A thick layer of polymer deposit was detected by ToF-SIMS and the results obtained by DSIMS measurements are in agreement with ToF-SIMS and XPS analyses and confirms that the coating synthesis was a success. The developed coating was active against both gram negative and gram positive tested bacteria. Conclusion The success of the chitosan immobilization was proven using the surface characterization techniques applied in this study. The coating was found to be effective against Escherichia coli and Staphylococcus aureus strains. © 2017 D'Almeida et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Binding of a highly de-N-acetylated chitosan to Japanese pheasant lysozyme (JPL), which differs from hen egg white lysozyme (HEWL) by nine amino acid substitutions (including Arg114-->His), was investigated by 1H-NMR spectroscopy. The profile of the one-dimensional spectrum of JPL is essentially identical to that of HEWL. Using two-dimensional spectra of JPL and HEWL, several aromatic and aliphatic proton resonances of JPL were assigned by comparison. When a highly de-N-acetylated chitosan (number-average degree of polymerization, about 18; degree of acetylation, 0.04), where the N-acetylated units are predominantly surrounded by de-N-acetylated units (a monoacetylated chitosan), was added to the JPL solution, the NMR signals were clearly affected in Trp28 C5H and Ile98 gammaCH, as in the case of binding to HEWL. The dissociation constant of the monoacetylated chitosan evaluated from the NMR signal responses was calculated to be 0.23+/-0.05 mm (-31.5 kJ/mol), which is similar to that of HEWL (0.11+/-0.02 mm, -33.3 kJ/mol). Thus, the Arg-->His substitution of the 114th amino acid, which participates in sugar residue binding at the right-sided subsite F, did not significantly affect the chitosan binding. In addition, the C2H signal of His114 of JPL was not affected by the chitosan binding. These results suggest that the monoacetylated chitosan binds to subsites E and F through the left-sided binding mode.

Polypropylene sutures (PP) are already used in surgery. Because microbial infection leads to complications, we developed antimicrobial PP suture by plasma-induced graft polymerization of acrylic acid followed by chitosan binding on the remaining carboxyl groups. Mechanical properties and surface morphologies were analyzed on these sutures. Tetracycline hydrochloride (TC) or nanosilver (NS) was then immobilized to PP. The resulting PP sutures evidenced drug release properties and antimicrobial activity in vitro. PP implanted in vivo for 30 days in the muscle of rats showed the absence of adverse effects and a tissue organization. This new polypropylene suture with suitable antimicrobial features appears to be a promising macromolecular material for clinical and cosmetic applications.

Optimization of the technological parameters affecting the mechanical properties and permeability of capsules is essential to produce capsules with improved properties for cell immobilization. In the present paper, the effect of different parameters on the technological properties of alginate/Ca2+/oligochitosan capsules has been investigated. The correct adjustment of the alginate concentration in the polymer matrix and the oligochitosan molar mass, concentration and coating time, have been found to be key parameters in obtaining porous and mechanically stable alginate/Ca2+/oligochitosan capsules. Results showed that an increase in the coating time and concentration of the alginate generated more stable capsules with a reduced membrane cut-off. Furthermore, we have established some correlations between capsule properties and the effectiveness of chitosan binding within the capsule's membrane. Data addressed herein could be a valid tool to fabricate optimized alginate/Ca2+/oligochitosan capsules with a potential for use in cell immobilization technology. Copyright © 2006 Society of Chemical Industry

The stability and controlled release of certain active substances (such as flavors, drugs, enzymes, essential oils, etc.) can be achieved through encapsulation. Ionically cross-linked hydrogels such as alginate- or alginate-chitosan-CaCl2 beads were proposed as suitable materials for encapsulation systems (Deladino et al. 2008; Han et al. 2008). Alginate beads coated with chitosan were used for encapsulation and release of different proteins (Coppi and Iannuccelli 2009; Zhou et al. 2010). Direct interaction between them forms beads with improved mechanical properties associated with reinforcement of bead structure due to chitosan binding to free alginate sites by cooperative ionic bounds (Deladino et al. 2008).

THE LIQUID-CRYSTALLINE STATE OF DNA The Condensed State of the High-Molecular-Mass Double-Stranded DNA The DNA Condensation and Aggregation Polyphosphates as a Simplified DNA Model Models of High-Molecular-Mass DNA Condensation in Water-Polymeric Solutions Grosberg Model of High-Molecular-Mass DNA Condensation Liquid-Crystalline Phases of the Low-Molecular-Mass Double-Stranded DNA Molecules Ordering of Low-Molecular-Mass Double-Stranded DNAs Brief Concept of Types of Liquid-Crystalline Phases Liquid-Crystalline Phases of Low-Molecular-Mass Double-Stranded DNA Molecules Dispersions of Low-Molecular-Mass Double-Stranded DNA Molecules Low-Molecular-Mass Double-Stranded DNA Dispersions in Water-Polymer Solutions Formation of DNA Dispersions in PEG-Containing Solutions Circular Dichroism of Nucleic Acid Dispersions Circular Dichroism as a Method of Proof of Cholesteric Packing of Nucleic Acid Molecules in Dispersion Particles and Analysis of Their Properties Effect of Different Factors on Formation and Properties of CLCD Particles Parameter of Nucleic Acid Molecule Order in CLCD Particles Polymorphism of Liquid-Crystalline Structures Formed by (DNA-Polycation) Complexes Some Peculiarities of Interaction of DNA Molecules with Polycations Specificity of Chitosan Binding to DNA Formation of Dispersions of (DNA-Chitosan) Complexes CD Spectra of Dispersions Formed by (DNA-Chitosan) Complexes X-Ray Parameters of Phases Formed by (DNA-Chitosan) Complexes Dependence of Efficiency of CLCD Formation by (DNA-Chitosan) Complexes on Various Factors Peculiarities of Interaction of Chitosan Molecules with Nucleic Acids Attempt at a Theoretical Description of Interactions Occurring in the (DNA-Chitosan) Complexes and Resulting in the Formation of Liquid-Crystalline Dispersions with Different Optical Properties Liquid-Crystalline State of DNA Circular Molecules Phase Exclusion of Circular Molecules of Nucleic Acids Formation of Dispersions From Circular Superhelical DNA CD Spectra of Circular Superhelical DNA Dispersions Under Conditions That Modify Parameters of Their Secondary Structure Packing Density and Rearrangement of the Spatial Structure of Superhelical DNA Molecules in LCD Particles Topological Forms and Rearrangement of the Spatial Organization of Superhelical DNA Molecules in LCD Particles DNA LIQUID-CRYSTALLINE FORMS AND THEIR BIOLOGICAL ACTIVITY Liquid-Crystalline State of DNA in Biological Objects DNA and Biological Objects DNA Reactions under Conditions Causing Liquid-Crystalline Dispersions Molecular Crowding Condensation of DNA under the Effect of Chitosan in Conditions Causing Molecular Crowding Activity of Nucleolytic Enzymes Under Conditions of Molecular Crowding Activity of Proteolytic Enzymes under Conditions of Molecular Crowding DNA LIQUID-CRYSTALLINE DISPERSIONS IN NANOTECHNOLOGY AND BIOSENSORICS Nanoconstructions Based on Nucleic Acid Molecules The General Concept of Nanotechnology Biological Molecules as a Background for Nanodesign Two Strategies of Nanodesign Based on NA Molecules Biosensors Based on Nucleic Acids General Concept of Construction and Operation of Biosensors Double-Stranded DNA Molecule as Polyfunctional Biosensing Unit Content and Principle of Operation of an Optical Biosensor Based on DNA Liquid-Crystalline Dispersions DNA CLCD Particles as Sensing Units Sandwich-Type Biosensing Units Based on (DNA-Polycation) Liquid-Crystalline Dispersions DNA Nanoconstruction as a Sensing Unit (New Type of Biodetectors) Hydrogels Containing DNA NaCs as New "Film-Type Biodetectors Index

Previously, we reported properties of a glycosylase belonging to GH-8 glycosyl hydrolase (GH) and having both chitosanase and glucanase activities. This enzyme (D2), whose molecular mass (86 kDa) was the largest among the GH-8 group, has its catalytic domain at the N-terminal region, and discoidin domain (DD) at the C-terminal region. Although various chitosanases, chitinases and glucanases have been known, DD is unique to the D2 enzyme. Glucanase and chitinase, but not chitosanase, are known to have functional domain such as carbohydrate-binding module, besides catalytic domain. Accordingly, function of the DD of D2 chitosanase was analyzed, using zygomycete cell wall containing chitosan, glucan and chitin as the basic constituents. The DD specifically and tightly bound to chitosan, but did not participate in affinity for glucan and chitin. Deletion of the DD caused marked reduction in absorbability to cell wall and in hydrolytic activity toward chitosan and glucan. These results suggest that the DD is concerned in binding of the enzyme to cell wall and in effective digestion of the insoluble substrate, through hydrolysis of not only chitosan but also coexisting glucan. Thus, this is the first example of chitosan-binding domain among various carbohydrate-binding modules reported thus far.