Lytic Polysaccharide Monooxygenase Activity of Tma12 Is Critical for Its Toxicity to Whitefly.

Lytic polysaccharide monooxygenases (LPMOs) are powerful redox enzymes that transform complex carbohydrates through oxidation and make them suitable for saccharification by canonical hydrolases. Due to this property, LPMOs are considered to be a valuable component of enzymatic consortia for industrial biorefineries. Tma12 is a fern entomotoxic protein that kills whitefly and has structural similarities with chitinolytic LPMO. However, its enzymatic activity is poorly understood. Studying the role of the LPMO-like activity in the insecticidal function of Tma12 can be of considerable importance. Our results show that Tma12 preferentially binds and digests ß-chitin. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis shows that the digestion of chitin produces chitin oligosaccharides of various lengths (DP2-DP7). The Michaelis constant (km) and catalytic constant (kcat) for hydrocoerulignone are 0.022 mM and 0.044 s-1, respectively. The attenuation of catalytic activity through diethylpyrocarbonate modification abolishes the insecticidal activity of the protein. Our findings reveal that (a) Tma12 is an active LPMO and (b) LPMO activity is indispensable for its function as a bioinsecticide.

The effect of chemical and structural modifiers on the haemostatic process and cytotoxicity of the beta-chitin patch.

Beta-chitin patch has previously been proven to be an effective haemostat, but whether modifying the patch affects its efficacy and safety, remains unanswered. In this study, the patch was modified using polyethylene oxide, Pluronic-F127, calcium, increased thickness or polyphosphate, and their effect on the process of haemostasis and cytotoxicity was tested and compared with standard-of-care, Surgicel and FloSeal. Whole blood collected from volunteers was applied to the patches to test their whole blood clotting and thrombin generation capacities, whilst platelet isolates were used to test their platelet aggregation ability. The fluid absorption capacity of the patches was tested using simulated body fluid. Cytotoxicity of the patches was tested using AlamarBlue assays and PC12 cells and the results were compared with the standard-of-care. In this study, beta-chitin patch modifications failed to improve its whole blood clotting, platelet aggregation and thrombin generation capacity. Compared to non-modified patch, modifications with polyethylene oxide or calcium reduced platelet aggregation and thrombin generation capacity, while increasing the thickness or adding polyphosphate decreased platelet aggregation capacity. The cytotoxicity assays demonstrated that the beta-chitin patches were non-toxic to cells. In vivo research is required to evaluate the safety and efficacy of the beta-chitin patches in a clinical setting.

Photocatalytic exoskeleton: Chitin nanofiber for retrievable and sustainable TiO2 carriers for the decomposition of various pollutants.

Loading a photocatalytic TiO2 to organic carriers has been desired for volumetric TiO2 incorporation, facile retrieval, and sustainable utilization. Traditionally, suspended TiO2 nanoparticles or its thin film on two-dimensional substrate are popularly fabricated for pollutants decomposition without carriers; due to poor thermomechanical properties of the organic carriers. Herein, a combination of the chitin nanofiber carrier and atomic layer deposition proves relevance for formation of anatase TiO2 thin layer so that photocatalytic decomposition in three-dimensional surface. Moreover, chitin nanofiber is capable of holding the TiO2 nanoparticles for multiple cycles of photocatalysis. Those types of TiO2 show characteristic degradation performance for gaseous (acetaldehyde) and aqueous pollutants (4-chlorophenol and rhodamine B). After catalytic reaction, chitin/TiO2 is retrievable owing to carrier's robustness even in water without TiO2 aggregation and loss. This work suggests that chitin-based photocatalyst is applicable to numerous pollutants through chitin's relatively high chemical resistance and stably wedged TiO2 during photocatalytic reaction.

Multifunctional chitin-based barrier membrane with antibacterial and osteogenic activities for the treatment of periodontal disease.

The guided tissue regeneration technique is an effective approach to repair periodontal defect. However, collagen barrier membranes used clinically lose stability easily, leading to soft tissue invasion, surgical site infection, and failure of osteogenesis. An ideal barrier membrane should possess proper antibacterial, osteoconductive activities, and favorable biodegradation. In this study, zinc oxide nanoparticles were homogeneously incorporated into the chitin hydrogel (ChT-1%ZnO) through one-step dissolution and regeneration method from alkaline/urea solution the first time. The remaining weights of ChT-1%ZnO in 150 µg/mL lysozyme solution was 52% after 5 weeks soaking. ChT-1%ZnO showed statistical antibacterial activities for P. gingivalis and S. aureus at 6 h, 12 h, and 24 h. Moreover, ChT-1%ZnO exhibits osteogenesis promotion in vitro, and it was further evaluated with rat periodontal defect model in vivo. The cemento-enamel junction value in ChT-1%ZnO group is 1.608 mm, presenting a statistical difference compared with no-membrane (1.825 mm) and ChT group (1.685 mm) after 8 weeks postoperatively.

Multi-functional carboxymethyl chitin-based nanoparticles for modulation of tumor-associated macrophage polarity.

Current challenge of using cytokines is its poor distribution and systemic side effects. To avoid this issue, we prepared the tumor-targeted and microenvironment-responsive nanocarriers (TRN), which were consisted of α-tocopheryl succinate (α-TOS) loaded mesoporous silica nanoparticles as cores, and surface-modified by thioketal-linkage, electrostatically coated with carboxymethyl chitin, and further anchored glucose-regulated protein 78-binding peptide as shells for encapsulating IL-12. TRN showed a size of 260 nm after encapsulated IL-12 and α-TOS with loading content of 0.0206% and 7.21%, respectively, and exhibited good biocompatibility to 4 T1 cells and macrophages. Moreover, IL-12/α-TOS loaded TRN displayed obvious anti-tumor efficacy on BALB/c nude mice bearing 4 T1 tumors, which was derived from promoted targeting to tumor tissue, endocytosed by macrophages and locally release IL-12 to subsequently repolarize tumor-associated macrophages into tumoricidal M1 phenotype with reduced side effects. The nanosystem exhibited as a promising strategy with functional conversion of macrophages in tumor microenvironment for anti-tumor therapy.

Alginate-chitosan oligosaccharide-ZnO composite hydrogel for accelerating wound healing.

Moist, breathable and antibacterial microenvironment can promote cell proliferation and migration, which is beneficial to wound healing. Here, we fabricated a novel sodium alginate-chitosan oligosaccharide­zinc oxide (SA-COS-ZnO) composite hydrogel by spontaneous Schiff base reaction, using aldehydated sodium alginate (SA), chitosan oligosaccharide (COS), and zinc oxide (ZnO) nanoparticles, which can provide a moist and antibacterial environment for wound healing. The porosity and swelling degree of SA-COS-ZnO hydrogel are 80% and 150%, respectively, and its water vapor permeability is 682 g/m2/24h. The composite hydrogel showed good biocompatibility to blood cells, 3T3 cells, and 293T cells, and significant antibacterial activity against Escherichia coli, Staphylococcus aureus, Candida albicans, and Bacillus subtilis. Moreover, the hydrogel showed a promoting effect on wound healing in a rat scald model. The present study suggests that marine carbohydrates composite hydrogels are promising in wound care management.

A Targeted Nano Drug Delivery System of AS1411 Functionalized Graphene Oxide Based Composites.

A novel method for the preparation of antitumor drug vehicles has been optimized. Biological materials of chitosan oligosaccharide (CO) and γ-polyglutamic acid (γ-PGA) have previously been employed as modifiers to covalently modify graphene oxide (GO), which in turn loaded doxorubicin (DOX) to obtain a nano drug delivery systems of graphene oxide based composites (GO-CO-γ-PGA-DOX). The system was not equipped with the ability of initiative targeting, thus resulting into toxicity and side effects on normal tissues or organs. In order to further improve the targeting property of the system, the nucleic acid aptamer NH2 -AS1411 (APT) of targeted nucleolin (C23) was used to conjugate on GO-CO-γ-PGA to yield the targeted nano drug delivery system APT-GO-CO-γ-PGA. The structure, composition, dispersion, particle size and morphology properties of the synthesized complex have been studied using multiple characterization methods. Drug loading and release profile data showed that APT-GO-CO-γ-PGA is provided with high drug loading capacity and is capable of controlled and sustained release of DOX. Cell experimental results indicated that since C23 was overexpressed on the surface of Hela cells but not on the surface of Beas-2B cells, APT-GO-CO-γ-PGA-DOX can target Hela cells and make increase toxicity to Hela cells than Beas-2B cells, and the IC50 value of APT-GO-CO-γ-PGA-DOX was 3.23±0.04 µg/mL. All results proved that APT-GO-CO-γ-PGA can deliver antitumor drugs in a targeted manner, and achieve the effect of reducing poison, which indicated that the targeted carrier exhibits a broad application prospect in the field of biomedicine.

Drug delivery system of dual-responsive PF127 hydrogel with polysaccharide-based nano-conjugate for textile-based transdermal therapy.

Pluronic F-127 based dual-responsive (pH/temperature) hydrogel drug delivery system was developed involving polysaccharide-based nano-conjugate of hyaluronic acid and chitosan oligosaccharide lactate and applied for loading of gallic acid which is the principal component of traditional Chinese medicine Cortex Moutan recommended in the treatment of atopic dermatitis. The polysaccharide-based nano-conjugate was used as pH-responsive compound in the formulation and its amphiphilic character was determined colorimetrically. Microstructure analysis by SEM and TEM indicated highly porous hydrogel network and well-dispersed micellar structures, respectively, after modification with the nano-conjugate, and so, release property of the hydrogel for drug was significantly improved. Different pH-conditions were applied here to see pH-responsiveness of the formulation and increase in acidity of external environment gradually diminished mechanical stability of the hydrogel and that was reflected on the drug release property. Rheology was performed to observe sol-gel transition of the formulation and showed better rheological properties after modification with nano-conjugate. In this study, the cytotoxicity results of PF127 based formulations loaded with/without gallic acid showed cell viability of > 80.0 % for human HaCaT keratinocytes in the concentration range of 0.0-20.0 µg/ml.

Thermosensitive and pH-responsive tannin-containing hydroxypropyl chitin hydrogel with long-lasting antibacterial activity for wound healing.

Polysaccharide hydrogels have been widely used as wound dressings because of their biocompatibility and ability to provide moist environment for wound healing. However, bacterial infection often delays the healing process. Herein, a novel thermosensitive and pH-sensitive hydroxypropyl chitin/tannic acid/ferric ion (HPCH/TA/Fe) composite hydrogel was fabricated by a simple assembly. The pre-cooled hydrogel precursor solution can be injected onto the irregular wound area and gel rapidly at physiological temperature. The TA not only acted as a crosslinker to enhance mechanical properties of the hydrogel, but also as an antibacterial agent which could be sustainably released in response to the acidic environment. The composite hydrogel showed excellent broad-spectrum antibacterial activity up to 7 days with negligible cytotoxicity. Moreover, the hydrogel can inhibit bacterial infection and accelerate the wound healing process without scars in the mouse experiment. These results indicate the potential application of this composite hydrogel for the infected wound healing.

Oligochitosan modified albumin as plasmid DNA delivery vector: Endocytic trafficking, polyplex fate, in vivo compatibility.

Cationic macromolecules condense DNA into small nanoparticles and form polyplex. The composition of the polyplex determines the endocytic process, the intracellular routing and the fate of the polyplex. Previously, oligochitosan-modified vectors with different protein moieties are used as gene delivery vector and the types of protein moiety can influence the endosome escape ability and transfection efficiency. Among the modified vectors, oligochitosan-modified bovine serum albumin (BSA) showed 90% transfection efficeincy compared to the modified zein and ovalbumin. These data encouraged us to investigate the mechanism of internalization involved in the superior transfection efficiency of modified BSA/ plasmid polyplex. The effect of specific endocytic inhibitors was studied in two adherent cell lines. The caveolae-mediated and lipid-mediated pathways play a significant role in the polyplex internalization. Next, a colocation of polyplex with lysosome was investigated in the presence of LysoTracker using confocal microscopy. Up to 70% of polyplex successfully escaped the lysosome without degradation. Four non-adherent cell lines showed above than 60% transfection efficiency at an optimized vector/plasmid ratio. Moreover, no significant hemolytic effect was observed up to 500 µg/mL of cationic BSA, indicating no detectable cell membrane disruption. Overall, the hybrid biomacromolecule showed good intracellular delivery and safety in a mice model.

The bioactivity of new chitin oligosaccharide dithiocarbamate derivatives evaluated against nematode disease (Meloidogyne incognita).

Plant-parasitic nematodes cause substantial crop losses annually; however, current nematicides are environmentally unfriendly and highly toxic to nontarget organisms. The development of green efficient nematicides from multifunctional natural bioactive substances such as chitin oligosaccharide (COS) is promising. In this paper, COS dithiocarbamate derivatives (COSDTC, COSDTA, COSDTB) were synthesized to increase nematicidal activity (against Meloidogyne incognita), and their structures were characterized by FTIR, NMR, TGA/DTG and elemental analysis. Furthermore, the nematicidal activities, egg hatching inhibitory activities, plant growth adjustment abilities, cytotoxicity and phytotoxicity of the derivatives were evaluated. The primary mechanism was assessed by heavy metal ion absorption and GSH-binding assays. The results showed COS dithiocarbamate derivatives could possess multiple efficacies, including high nematicidal activities and egg hatching inhibitory activities, plant growth regulating effects, low cell toxicities and phytotoxicities. Additionally, it was inferred that nematicidal activity may be correlated with GSH-binding activity but not heavy metal ion complexation. COS modification has immense potential for controlling plant-parasitic nematodes.

Preparation, Mechanical Properties, and Biocompatibility of Graphene Oxide-Reinforced Chitin Monofilament Absorbable Surgical Sutures.

Chitin (CT) is a good material to prepare surgical sutures due to its conspicuous biological characteristics. However, the poor mechanical strength of pure CT sutures limits its application. In order to improve its strength, a composite monofilament absorbable suture was prepared in this study using graphene oxide and chitin (GO-CT) using a green method. FT-IR spectra showed that GO-CT contained the characteristic functional groups of GO and CT, indicating that a GO-CT suture was successfully obtained. With the addition of a small amount of GO (1.6wt% solution) in chitin, the breaking tensile strength, knot strength, and knot-pull strength of the GO-CT suture were significantly improved compared to the CT suture. The biocompatibility of the GO-CT suture in vitro was checked by tetrazolium-based colorimetric assays and no cytotoxicity to L929 cells was found. In vivo, the subcutaneous implantation of GO-CT sutures in the dorsal skin of rats found no abnormalities by hematoxylin-eosin staining. Furthermore, there were no significant changes in the gene expression of the inflammatory mediators, interleukin 1ß (IL-1ß), tumor necrosis factor-α, IL-6, IL-17A, interferon-γ, or IL-10; however, the expression of transforming growth factor ß was significantly increased in the first week. In summary, GO-CT sutures may have potential as a suture material in the clinic.

Linolenic acid-modified methoxy poly (ethylene glycol)-oligochitosan conjugate micelles for encapsulation of amphotericin B.

Introduction of linolenic acid (LNA) and methoxy poly (ethylene glycol) (MPEG) to the backbone of oligochitosan (CS) afforded LNA-modified MPEG-CS conjugate (MPEG-CS-LNA). Amphotericin B-loaded MPEG-CS-LNA micelles (AmB-M) were prepared via dialysis method with 82.27 ± 1.96% of drug encapsulation efficiency and 10.52 ± 0.22% of drug loading capacity. The AmB-M enhanced AmB's water-solubility to 1.64 mg/mL, being 1640-folds higher than native AmB. The AmB-M obviously reduced hemolytic effect and renal toxicity of AmB when compared to marketed AmB injection (AmB-I). Its antifungal activity against Candida albicans was equivalent to AmB-I although AmB's release from AmB-M was significantly retarded. According to fluorescence microscopy test, the unchanged activity should be attributed to enhanced fungal cellular uptake of AmB-M caused by combined inducement of LNA and CS. The pharmacokinetic studies demonstrated that AmB-M also improved the pharmacokinetic parameters of AmB with AmB-I as control. Conclusively, developed LNA-modified MPEG-CS micellar system could be a viable alternative to the current toxic commercial AmB-I as a highly efficacious drug delivery system.

A simple mechanical agitation method to fabricate chitin nanogels directly from chitin solution and subsequent surface modification.

The development of physical approaches and green technologies to construct novel chitin materials is essential for the exquisite utilization of the renewable and valuable resource of chitin. In the present study, chitin nanogels were simply fabricated from a chitin solution dissolved in 8% NaOH/4% urea aqueous solvent by high speed stirring. The mechanical stirring generated in situ heat that induced the regeneration of chitin chains and ensured good dispersion of the nanogels. The prepared nanogels were composed of spherical nanoparticles of size 20 to 30 nm with some aggregates. The formation of chitin nanogels was confirmed to be a physical process without using organic solvent or chemical crosslinking. Rheological tests revealed a shear thinning behavior of the nanogels and injectable hydrogels were developed accordingly. The chitin nanogels showed no toxicity to L929 cells and cell attachment on the surface of the nanogel was observed. Further, monodispersed cationic nanogels and anionic nanogels were facilely obtained by deacetylating and TEMPO-mediated oxidizing chitin nanogels, and demonstrated different antibacterial properties.

A hispanolone-derived diterpenoid inhibits M2-Macrophage polarization in vitro via JAK/STAT and attenuates chitin induced inflammation in vivo.

Macrophages are highly plastic cells that adopt different functional phenotypes in response to environmental signals. Classically activated macrophages (M1) exhibit a pro-inflammatory role, mediating host defense against microorganisms or tumor cells; whereas alternatively activated macrophages (M2) perform a range of physiological processes, including inflammation, wound repair and tissue remodeling. Interestingly, M2 macrophages have been involved in pathological settings such as tumor progression, parasitic infection and respiratory disorders. Consequently, the search of new agents able to control macrophage polarization is on the basis of new therapeutic strategies. In the present study, we have evaluated the effect of the hispanolone derivative 8,9-dehydrohispanolone-15,16-lactol (DHHL) on M2 macrophage polarization. Our results reveal that DHHL significantly inhibited IL-4- or IL-13-stimulated M2 macrophage activation, as showed by reduced expression of M2 markers. In addition, DHHL suppressed IL-4-induced STAT-6 and JAK-1 tyrosine phosphorylation, suggesting that this compound inhibited M2 polarization by suppressing the JAK-STAT signaling pathway. Finally, DHHL prevented eosinophil recruitment and the presence of F4/80+-CD206+ M2-like macrophages in an in vivo model of M2 polarization via administration of chitin. Collectively, these results confirm DHHL as a novel regulator of macrophage polarization suitable to design future therapies towards M2-macrophages mediated pathologies.

Spontaneous Chitin Accumulation in Airways and Age-Related Fibrotic Lung Disease.

The environmentally widespread polysaccharide chitin is degraded and recycled by ubiquitous bacterial and fungal chitinases. Although vertebrates express active chitinases from evolutionarily conserved loci, their role in mammalian physiology is unclear. We show that distinct lung epithelial cells secrete acidic mammalian chitinase (AMCase), which is required for airway chitinase activity. AMCase-deficient mice exhibit premature morbidity and mortality, concomitant with accumulation of environmentally derived chitin polymers in the airways and expression of pro-fibrotic cytokines. Over time, these mice develop spontaneous pulmonary fibrosis, which is ameliorated by restoration of lung chitinase activity by genetic or therapeutic approaches. AMCase-deficient epithelial cells express fibrosis-associated gene sets linked with cell stress pathways. Mice with lung fibrosis due to telomere dysfunction and humans with interstitial lung disease also accumulate excess chitin polymers in their airways. These data suggest that altered chitin clearance could exacerbate fibrogenic pathways in the setting of lung diseases characterized by epithelial cell dysfunction.

Studies on electrochemical glucose sensing, antimicrobial activity and cytotoxicity of fabricated copper nanoparticle immobilized chitin nanostructure.

In this study, copper nanoparticle immobilized chitin nanocomposite (CNP/CuNP) was synthesized and used for the development of non-enzymatic electrochemical sensor. The CNP/CuNP was characterized by X-ray diffraction (XRD), fourier transform infra red (FTIR) spectroscopy and high resolution transmission electron microscopy (HRTEM) analysis. The glucose sensing property of CNP/CuNP was investigated by cyclic voltammetry (CV) and chronoamperometry (CA). As a result of the synergistic effect of CNP and CuNP, the modified electrode displayed effective electro-oxidation of glucose in 0.1M NaOH solution. At 0.45V potential the modified electrode showed response towards glucose in the linear range of 1-1000µM with a lowest detection limit of 0.776µM with better selectivity and stability. In addition, the antimicrobial activity of CNP/CuNP was evaluated against bacterial and fungal strains. CNP/CuNP displayed enhanced antimicrobial activity when compared to CNP and CuNP alone. Similarly, cytotoxicity of CNP/CuNP was tested against Artemia salina, which showed no toxic effect in the tested concentration.

Homogeneous synthesis of quaternized chitin in NaOH/urea aqueous solution as a potential gene vector.

Water-soluble quaternized chitins (QCs) were homogeneously synthesized by reacting chitin with (3-chloro-2-hydroxypropyl) trimethylammonium chloride (CHPTAC) in 8wt% NaOH/4wt% urea aqueous solutions. The chemical structure and solution properties of the quaternized chitins were characterized by (1)H NMR, FT-IR, elemental analysis, dynamic light scattering (DLS) and zeta potential measurements. The results demonstrated that the water-soluble QCs, with a degree of substitution (DS) values of 0.27-0.54, could be obtained by varying the concentration of chitin, the molar ratio of CHPTAC to chitin unit, and the reaction time at room temperature (25°C). Two QCs (DS=0.36 and 0.54) were selected and studied as gene carriers. Agarose gel retardation assay revealed that both QCs could condense DNA efficiently when N/P ratio>3. The results of particle size and zeta potential indicated that both QCs had a good ability of condensing plasmid DNA into compact nanoparticles with the size of 100-200nm and zeta potential of +18 to +36mV. Compared to polyethylenimine (PEI, 25kDa), the QCs exhibited outstanding low cytotoxicity. Transfection efficiencies of the QCs/DNA complexes were measured using pGL-3 encoding luciferase as the foreign DNA, and the QCs/DNA complexes showed effective transfection efficiencies in 293T cells. These results revealed that the QCs prepared in NaOH/urea aqueous solutions could be used as promising non-viral gene carriers owing to their excellent characteristics.

Thermosensitive injectable in-situ forming carboxymethyl chitin hydrogel for three-dimensional cell culture.

Injectable hydrogels have gained great attentions for cell therapy and tissue regeneration as a result of the applications in minimally invasive surgical procedures with the ease of handling and complete filling of the defect area. Here, a novel biodegradable, thermosensitive and injectable carboxymethyl chitin (CMCH) hydrogel was developed for three-dimensional (3D) cell culture. The obtained CMCH solution remained transparent liquid flowing easily at low temperatures and gelled rapidly at 37°C. The gelation time of CMCH hydrogels could be easily tuned by varying temperature and the degree of carboxymethylation, which facilitates the cell encapsulation process at room temperature and in-situ forming hydrogel at body temperature. Moreover, the CMCH-14 hydrogels in PBS buffer remained stable and continuous porous structure and could be degraded in the presence of lysozyme or hyaluronidase. HeLa cells proliferated sustainably and self-assembled to form 3D multicellular spheroids with high cell activity on the surface of CMCH-14 hydrogel. Encapsulation of COS-7 cells within the in-situ forming CMCH hydrogel demonstrated that CMCH hydrogels promoted cell survival and proliferation. In vivo mouse study of the CMCH hydrogels showed good in-situ gel formation and tissue biocompatibility. Thus, the biodegradable thermosensitive injectable CMCH hydrogels hold potential for 3D cell culture and biomedical applications. STATEMENT OF SIGNIFICANCE: Biodegradable hydrogels have been widely studied for cell therapy and tissue regeneration. Herein, we report a novel thermosensitive injectable carboxymethyl chitin (CMCH) hydrogel for 3D cell culture, which was synthesized homogeneously from the bioactive natural chitin through the "green" process avoiding using organic solvent. The CMCH solutions exhibited rapid thermoresponsive sol-to-gel phase transition behavior at 37°C with controllable gelation times, which facilitates the cell encapsulation process at room temperature and in-situ forming hydrogel at body temperature. Importantly, in vitro 3D cell culture and in vivo mouse study of the CMCH hydrogel showed promotion of cell survival and proliferation, good in-situ gel formation and biocompatibility. We believe that such thermosensitive injectable CMCH hydrogels would be very useful for biomedical applications, such as tumor model for cancer research, post-operative adhesion prevention, the regeneration of cartilage and central nervous system and so on.

Antimicrobial and antitumor activities of chitosan from shiitake stipes, compared to commercial chitosan from crab shells.

Chitosan was prepared by alkaline N-deacetylation of chitin obtained from shiitake stipes and crab shells and its antimicrobial and antitumor activities were studied. Chitosan from shiitake stipes and crab shells exhibited excellent antimicrobial activities against eight species of Gram positive and negative pathogenic bacteria with inhibition zones of 11.4-26.8mm at 0.5mg/ml. Among chitosan samples, shiitake chitosan C120 was the most effective with inhibition zones of 16.4-26.8mm at 0.5mg/ml. In addition, shiitake and crab chitosan showed a moderate anti-proliferative effect on IMR 32 and Hep G2 cells. At 5mg/ml, the viability of IMR 32 cells incubated with chitosan was 68.8-85.0% whereas that of Hep G2 cells with chitosan was 60.4-82.9%. Overall, shiitake chitosan showed slightly better antimicrobial and antitumor activities than crab chitosan. Based on the results obtained, shiitake and crab chitosan were strong antimicrobial agents and moderate antitumor agents.