Showing papers on "Chitin published in 2002"

Chitin and Chitosan

Abstract: The article contains sections titled: 1. Introduction 2. Molecular Structure and Conformation 2.1. Chitin 2.2. Chitosan 3. Raw Materials and Production 3.1. Isolation of Chitin from Crab and Shrimp Shells 3.2. Preparation of Chitosan from Chitin 4. Metabolism and Biosynthesis 5. Chemical Properties 5.1. Reactions on the Amino Group 5.1.1. N-Acylation 5.1.2. Formation of N-Alkylidene and N-Arylidene Derivatives 5.1.3. N-Alkylation and N-Arylation 5.2. Reactions at the Hydroxyl Group 5.3. Reactions at C-6 5.4. Graft Polymerization on Chitin and Chitosan 6. Application Forms and Formulations 7. Uses 8. Economic Aspects 9. Toxicology and Environmental Aspects

Antimicrobial activity of shrimp chitin and chitosan from different treatments and applications of fish preservation.

Abstract: The effects of the deacetylation degree (DD) and preparation methods for chitosan on antimicrobial activity were evaluated Chemically prepared chitin (CH-chitin) and microbiologically prepared chitin (MO-chitin) were obtained from shrimp shells The CH-chitin and MO-chitin were further chemically deacetylated to obtain various chitosan products of which their DD ranged from low (47–53%) through medium DD (74–76%) to high (95–98%) In addition, MO-chitin was deacetylated also by various proteases The antimicrobial activities of these products were evaluated in medium with pH 60 Neither the CH-chitin, MO-chitin nor protease-deacetylated chitinous products showed any antimicrobial activity For chitosan, antimicrobial activity increased with increasing DD, and was stronger against bacteria than against fungi The minimal lethal concentrations (MLC) of chitosan with a high DD against Bacillus cereus, Escherichia coli, Listeria monocytogenes, Pseudomonas aeruginosa, Shigella dysenteriae, Staphylococcus aureus, Vibrio cholerae, and V parahaemolyticus were all in the range of 50–200 ppm, whereas the MLC against Candida albicans and Fusarium oxysporum were 200 ppm and 500 ppm, respectively No antifungal activity was found at 2000 ppm against Aspergillus fumigatus or A parasiticus Pretreatment of fish fillets (Oncorhynchus nereka) with 1% chitosan solution (high DD) for 3 h retarded the increase in the volatile basic nitrogen content, as well as the counts for mesophiles, psychrotrophs, coliforms, Aeromonas spp, and Vibrio spp The shelf life was consequently extended from 5 days to 9 days

The genetic complexity of chitin synthesis in fungi.

Abstract: Chitin synthesis is a process maintained across the fungal kingdom that, thanks to the power of genetic manipulation of yeast cells, is now beginning to be understood. Chitin synthesis is based on the regulation of distinct chitin synthase isoenzymes whose number ranges from one in Schizosaccharomyces pombe to seven in some filamentous fungi, such as Aspergillus fumigatus. This high diversity makes it difficult to find a unique model of regulation. However, the results available suggest common themes in regulation. The arrival of the genomic era, together with the development of fungal genetic technology should allow experimental approaches to this process.

Developmentally regulated conversion of surface‐exposed chitin to chitosan in cell walls of plant pathogenic fungi

Abstract: Summary • Conversion of surface-exposed chitin to chitosan in cell walls of in vitro - and in vivo -differentiated infection structures of two rust fungi, the wheat stem rust fungus Puccinia graminis f. sp. tritici and the broad bean rust fungus Uromyces fabae , and of the causal agent of maize anthracnose, Colletotrichum graminicola , were studied. • Epi-fluorescence microscopy with the fluorescence-labeled lectin wheat germ agglutinin (WGA) revealed that surfaces of infection structures formed on the plant cuticle expose chitin, whereas surfaces of structures formed after invading the host do not. • To identify chitin modification by de- N -acetylation, we raised polyclonal antibodies specifically recognizing de- N -acetylated chitosan. These antibodies labeled only those infection structures that differentiate inside the plant, indicating that chitosan is exposed on cell wall surfaces post penetration. • Surface modification of the fungal cell walls by chitin de- N -acetylation is discussed as a fungal strategy to protect cell walls of pathogenic hyphae from enzymatic hydrolysis by host chitinases, and to avoid generation of an auto-catalytic defense response system in the invaded host tissue.

Chitin and chitosan for versatile applications

Abstract: Chitin and chitosan are versatile polymers, where the interest in chitosan is due to the large variety of useful forms that are commercially available or can be made available. Chitin basically is obtained from prawn/crab shells; chemical treatment of chitin produces chitosan. This article surveys applications of chitin and chitosan in various industrial and biomedical fields.

Chitinases A, B, and C1 of Serratia marcescens 2170 produced by recombinant Escherichia coli: enzymatic properties and synergism on chitin degradation.

Abstract: To discover the individual roles of the chitinases from Serratia marcescens 2170, chitinases A, B, and C1 (ChiA, ChiB, and ChiC1) were produced by Escherichia coli and their enzymatic properties as well as synergistic effect on chitin degradation were studied. All three chitinases showed a broad pH optimum and maintained significant chitinolytic activity between pH 4 and 10. ChiA was the most active enzyme toward insoluble chitins, but ChiC1 was the most active toward soluble chitin derivatives among the three chitinases. Although all three chitinases released (GlcNAc)2 almost exclusively from colloidal chitin, ChiB and ChiC1 split (GlcNAc)6 to (GlcNAc)3, while ChiA exclusively generated (GlcNAc)2 and (GlcNAc)4. Clear synergism on the hydrolysis of powdered chitin was observed in the combination between ChiA and either ChiB or ChiC, and the sites attacked by ChiA on the substrate are suggested to be different from those by either ChiB or ChiC1.

Chitin production by arthropods in the hydrosphere

Abstract: Chitin is widely distributed in nature and its annual production is thought to be huge. However, the chitin production has been rarely estimated in aquatic ecosystems, despite the growing economic interest in this polymer. Arthropods are one of the main chitin producers in the hydrosphere and a correct evaluation of the chitin production by these organisms in the different marine and freshwater ecosystems is of prime interest to understand their importance in the biogeochemical cycles of carbon and nitrogen. Such evaluation is also worth considering to achieve a rational exploitation of crustaceans which are currently the major source of chitin for the industry. Annual chitin production of crustaceans and insects in aquatic ecosystems was estimated on the basis of annual tissue production estimates and body chitin content measurements. About 800 annual tissue production estimates were collected from the literature. Estimates mainly concerned continental fresh waters and neritic ecosystems. Data were almost inexistent for athalassohaline and oceanic ecosystems. On the whole, 60% of the production estimates fell between 0.1 and 10.0 g dry weight m−2 yr−1. Published chitin levels in crustaceans and insects ranged from 3 to 16% of the whole body dry weight. Data were, however, lacking for some major groups such as trichopterans or amphipods. Aquatic insects and crustaceans were therefore collected and assayed for chitin using a highly specific enzymatic method. The chitin content of the collected insects (Coleoptera, Diptera, Ephemeroptera, Odonata, Plecoptera, Trichoptera) varied from 3 to 10% of the whole body dry weight; that of the collected crustaceans (Amphipoda, Branchiopoda, Copepoda) from 2.5 to 8.5% of the whole body dry weight. Total annual chitin production by arthropods had been estimated to 28 × 106 T chitin yr−1 for the freshwater ecosystems, to 6 × 106 T chitin yr−1 for athalassohaline ecosystems and to 1328 × 106 T chitin yr−1 for marine ecosystems. The importance of the chitin production corresponding to the formation of exuviae and peritrophic membranes in arthropods and the chitin production by non-arthropod organisms in the chitin budget of aquatic ecosystems was highlighted and discussed.

Microarray analysis of chitin elicitation in Arabidopsis thaliana

Abstract: Summary Chitin oligomers, released from fungal cell walls by endochitinase, induce defence and related cellular responses in many plants. However, little is known about chitin responses in the model plant Arabidopsis. We describe here a large-scale characterization of gene expression patterns in Arabidopsis in response to chitin treatment using an Arabidopsis microarray consisting of 2375 EST clones representing putative defence-related and regulatory genes. Transcript levels for 71 ESTs, representing 61 genes, were altered three-fold or more in chitin-treated seedlings relative to control seedlings. A number of transcripts exhibited altered accumulation as early as 10 min after exposure to chitin, representing some of the earliest changes in gene expression observed in chitin-treated plants. Included among the 61 genes were those that have been reported to be elicited by various pathogen-related stimuli in other plants. Additional genes, including genes of unknown function, were also identified, broadening our understanding of chitin-elicited responses. Among transcripts with enhanced accumulation, one cluster was enriched in genes with both the W-box promoter element and a novel regulatory element. In addition, a number of transcripts had decreased abundance, encoding several proteins involved in cell wall strengthening and wall deposition. The chalcone synthase promoter element was identified in the upstream regions of these genes, suggesting that pathogen signals may suppress the expression of some genes. These data indicate that Arabidopsis should be an excellent model to elucidate the mechanisms of chitin elicitation in plant defence.

Characterization of early, chitin-induced gene expression in Arabidopsis.

Abstract: Three genes (i.e., a zinc finger protein, a lectin-like protein, and AtMPK3), previously shown to respond to chitin elicitation in microarray experiments, were used to examine the response of Arabi...

Immunolocalization of chitin synthase in the tobacco hornworm

Abstract: To start investigation of chitin synthesis and peritrophic membrane formation in the midgut of Manduca sexta, we have cloned a cDNA fragment encoding chitin synthase. Northern blots with a corresponding RNA probe revealed a single transcript of 4.7 kb, which was most prominent in poly(A) RNA isolated from the anterior and median midgut as well as from tracheal cells. In situ hybridization showed that the amount of chitin synthase transcripts in the cytoplasm of columnar cells decreased from the anterior to the posterior midgut. Moreover, in the anterior midgut they were localized in the apical region of columnar cells. Southern blots suggested more than one gene locus for chitin synthase in the Manduca genome. To analyze the distribution of chitin synthases on the protein level, we expressed a polymerase chain reaction (PCR) fragment of 119 amino acids in Escherichia coli and generated polyclonal antibodies to the purified recombinant protein. In immunoblots of crude extracts derived from the anterior midgut as well as from partially purified brush border membranes of columnar cells the affinity-purified anti-chitin synthase antiserum labeled a single protein with an apparent molecular mass of 150–200 kDa. Immunohistochemistry showed intense labeling in midgut brush border membranes. Immunofluorescence was restricted to the apical ends of microvilli. Apical membranes of salivary glands and tracheal cells were labeled as well, but not those of Malpighian tubules. This is the first time that chitin synthase expression has been visualized in insect tissues on the level of proteins.

Antibacterial properties of chitosan in waterborne pathogen

Abstract: The antimicrobial properties of chitosan, a derivative of chitin, were investigated in the solid and liquid culture against bacteria associated with waterborne disease in order to assess the potential for using chitosan as a natural disinfectant. Six strains which included three gram-negative and three gram-positive bacteria were studied. The effects of the deacetylation degree, concentration, and molecular weight of chitosan on antibacterial activities were assessed. Chitosan exhibited the highest antibacterial activity against the Pseudomonas aeruginosa on the solid agar. Similar tendency was found when the bacteria were cultivated in liquid broth. The higher deacetylation degree and higher concentration of chitosan cause the higher antibacterial activities. The effect of molecular weight of chitosan on the inhibition efficacy of bacteria is dependent on the species of bacteria. Escherichia coli is sensitive to chitosan during its death phase and logarithmic phase. The antibacterial mechanism of chitosan was illustrated by the surface charge and persistence length. Results indicated that chitosan is potential as a natural disinfectant.

Involvement of GFA1, which encodes glutamine–fructose‐6‐phosphate amidotransferase, in the activation of the chitin synthesis pathway in response to cell‐wall defects in Saccharomyces cerevisiae

Abstract: Cell-wall damage caused by mutations of cell-wall-related genes triggers a compensatory mechanism which eventually results in hyperaccumulation of chitin reaching 20% of the cell-wall dry mass. We show that activation of chitin synthesis is accompanied by a rise, from 1.3-fold to 3.5-fold according to the gene mutation, in the expression of most of the genes encoding enzymes of the chitin metabolic pathways. Evidence that GFA1, which encodes glutamine–fructose-6-Phosphate amidotransferase (Gfa1p), the first committed enzyme of this pathway, plays a major role in this process was as follows. Activation of chitin synthesis in the cell-wall mutants correlated with activation of GFA1 and with a proportional increase in Gfa1p activity. Overexpression of GFA1 caused an approximately threefold increase in chitin in the transformed cells, whereas chitin content was barely affected by the joint overexpression of CHS3 and CHS7. Introduction of a gfa1-97 allele mutation in the cell-wall-defective gas1Δ mutant or cultivation of this mutant in a hyperosmotic medium resulted in reduction in chitin synthesis that was proportional to the decrease in Gfa1p activity. Finally, the stimulation of chitin production was also accompanied by an increase in pools of fructose 6-Phosphate, a substrate of Gfa1p. In quantitative terms, we estimated the flux-coefficient control of Gfa1p to be in the range of 0.90, and found that regulation of the chitin metabolic pathway was mainly hierarchical, i.e. dominated by regulation of the amount of newly synthesized GFA1 protein. In the search for the mechanism by which GFA1 is activated in response to cell-wall perturbations, we could only show that neither MCM1 nor RLM1, which encode two transcriptional factors of the MADS box family that are required for expression of cell-cycle and cell-wall-related genes, was involved in this process.

Antagonism of Bacillus species (strain BC121) towards Curvularia lunata.

Abstract: A soil bacterium, Bacillus sp. strain BC121, isolated from the rhizosphere of sorghum, showed high antagonistic activity against Curvularia lunata. A clear inhibition zone of 0.5-1 cm was observed in dual plate assay. After 10 days of incubation, the bacterial strain grew over the fungal mycelial surface and multiplied extensively on it. Scanning electron microscopic observations showed a clear hyphal lysis and degradation of fungal cell wall. In dual cultures, the Bacillus strain BC121 inhibited the C. lunata up to 60% in terms of dry weight. This strain also produced a clear halo region on chitin agar medium plates containing 0.5% colloidal chitin, indicating that it excretes chitinase. The role of the Bacillus strain BC121 in suppressing the fungal growth in vitro was studied in comparison with a mutant of that strain, which lacks both antagonistic activity and chitinolytic activity. The extra-cellular protein precipitate from Bacillus strain BC121 culture filtrate had significant growth-retarding effect and mycolytic activity on C. lunata. The protein extract from the wild strain, when tested on SDS-PAGE gel showed a unique band corresponding to the molecular mass of 25 kDa, which could be the probable chitinase protein.

Antifungal Activity of Rye (Secale cereale) Seed Chitinases: the Different Binding Manner of Class I and Class II Chitinases to the Fungal Cell Walls

Abstract: The antifungal activities of rye seed chitinase-a (RSC-a, class I) and -c (RSC-c, class II) were studied in detail using two different bioassays with Trichoderma sp. as well as binding and degradation experiments with the cell walls prepared from its mycelia. RSC-a inhibited more strongly the re-extension of the hyphae, containing mainly mature cells, than RSC-c did. Upon incubation of the fungus with fluorescent chitinases, FITC-labeled RSC-a was found to be located in the hyphal tips, lateral walls, and septa, while FITC-labeled RSC-c was only in the hyphal tip. RSC-a had a greater affinity for the cell walls than RSC-c. RSC-a liberated a larger amount of reducing sugar from the cell walls than RSC-c did. These results inferred that RSC-a first binds to the lateral walls and septa, consisting of the mature cell walls, and degrades mature chitin fiber, while RSC-c binds only to the hyphal tip followed by degradation of only nascent chitin. As a result, RSC-a inhibited fungal growth more effectively than RSC-c. Furthermore, it was suggested that the chitin-binding domain in RSC-a assists the antifungal action of RSC-a by binding to the fungal hypha.

Functional analysis of the chitin-binding domain of a family 19 chitinase from Streptomyces griseus HUT6037: substrate-binding affinity and cis-dominant increase of antifungal function.

Abstract: Chitinase C (ChiC) is the first bacterial family 19 chitinase discovered in Streptomyces griseus HUT6037. While it shares significant similarity with the plant family 19 chitinases in the catalytic domain, its N-terminal chitin-binding domain (ChBD(ChiC)) differs from those of the plant enzymes. ChBD(ChiC) and the catalytic domain (CatD(ChiC)), as well as intact ChiC, were separately produced in E. coli and purified to homogeneity. Binding experiments and isothermal titration calorimetry assays demonstrated that ChBD(ChiC) binds to insoluble chitin, soluble chitin, cellulose, and N-acetylchitohexaose (roughly in that order). A deletion of ChBD(ChiC) resulted in moderate (about 50%) reduction of the hydrolyzing activity toward insoluble chitin substrates, but most (about 90%) of the antifungal activity against Trichoderma reesei was abolished by this deletion. Thus, this domain appears to contribute more importantly to antifungal properties than to catalytic activities. ChBD(ChiC) itself did not have antifungal activity or a synergistic effect on the antifungal activity of CatD(ChiC) in trans.