Chitin synthesis in larval and pupal epidermis of the Indian meal moth,Plodia interpunctella (Hübner), and the greater wax moth,Galleria mellonella (L.)

The level of chitin synthesis was determined in whole last-instar larvae and in pupae of Plodia interpunctella, and in epidermal tissue from similar stages of Galleria mellonella. The incorporation of radiolabelled N-acetyl-d-glucosamine into chitin was used to measure synthesis. Chitin production was similar in both species with peaks of synthesis occurring at the beginning of the last larval instar, in prepupae, in white pupae and prior to adult emergence. P. interpunctella also exhibited an additional small increase at mid-instar. Exposure of larval epidermis of P. interpunctella to 20-hydroxyecdysone in vitro stimulated chitin synthesis, but only after a 24 hr lag period subsequent to exposure to the hormone. This hormonal stimulation of chitin synthesis was inhibited by actinomycin-D and cycloheximide which suggested that 20-hydroxyecdysone-stimulated production of chitin depended on synthesis of RNA and protein. Comparison of the synthesis of chitin in epidermis of G. mellonella with previously published ecdysone titres, indicated that chitin production in vivo is preceded by an elevated ecdysone titre.     

Role of Chitin-Binding Proteins in the Specific Attachment of the Marine Bacterium Vibrio harveyi to Chitin

We examined the mechanism of attachment of the marine bacterium Vibrio harveyi to chitin. Wheat germ agglutinin and chitinase bind to chitin and competitively inhibited the attachment of V. harveyi to chitin, but not to cellulose. Bovine serum albumin and cellulase do not bind to chitin and had no effect on bacterial attachment to chitin. These data suggest that this bacterium recognizes specific attachment sites on the chitin particle. The level of attachment of a chitinase-overproducing mutant of V. harveyi to chitin was about twice as much as that of the uninduced wild type. Detergent-extracted cell membranes inhibited attachment and contained a 53-kDa peptide that was overproduced by the chitinase-overproducing mutant. Three peptides (40, 53, and 150 kDa) were recovered from chitin which had been exposed to membrane extracts. Polyclonal antibodies raised against extracellular chitinase cross-reacted with the 53- and 150-kDa chitin-binding peptides and inhibited attachment, probably by sterically hindering interactions between the chitin-binding peptides and chitin. The 53- and 150-kDa chitin-binding peptides did not have chitinase activity. These results suggest that chitin-binding peptides, especially the 53-kDa chitin-binding peptide and chitinase and perhaps the 150-kDa peptide, mediate the specific attachment of V. harveyi to chitin.     

Serratia marcescens chitinase: One-step purification and use for the determination of chitin

The extracellular chitinase produced by Serratia marcescens was obtained in highly purified form by adsorption-digestion on chitin. After gel electrophoresis in a nondenaturing system, the purified preparation exhibited two major protein bands that coincided with enzymatic activity. A study of the enzyme properties showed its suitability for the analysis of chitin. Thus, the chitinase exhibited excellent stability, a wide pH optimum, and linear kinetics over a much greater range than similar enzymes from other sources. The major product of chitin hydrolysis was chitobiose, which was slowly converted into free N-acetylglucosamine by traces of β-N-acetylglucosaminidase present in the purified preparation. The preparation was free from other polysaccharide hydrolases. Experiments with radiolabeled yeast cell walls showed that the chitinase was able to degrade wall chitin completely and specifically.     

Comparison of physicochemical properties of chitins isolated from an insect (Melolontha melolontha) and a crustacean species (Oniscus asellus)

The chitin structures of two common European species belonging to Insecta (Melolontha melolontha) and Crustacea (Oniscus asellus) were isolated. The same procedure is followed for chitin isolations for both the species. First, HCl was used for removing of minerals in the organisms, and then, the protein structure was removed by using NaOH. Chitins obtained from these two species were characterized physicochemically. Physicochemical properties of chitins isolated from the insect and the crustacean were compared to each other. The chitin content for dry weights of M. melolontha and O. asellus were recorded as 13–14 and 6–7 %, respectively. The results of Fourier transform infrared spectroscopy, thermogravimetric analysis and X-ray diffraction analysis were found to be more or less similar. The surface morphologies of chitins were examined via environmental scanning electron microscopy and nanofibers, and pore structures were observed. While the chitin nanofibers of O. asellus were adherent to each other, nanofibers of M. melolontha were non-adherent. On the other hand, the number of pores was much higher in the chitin from M. melolontha than in the chitin from O. asellus. Looking at the elemental analysis results, the M. melolontha chitin was found to be more pure than the O. asellus chitin. For this reason, M. melolontha has been considered more attractive source for chitin than O. asellus.     

Preparation and physical properties of chitin fatty acids esters

Trifluoroacetic anhydride is an effective promoter for the preparation of chitin single- and mixed-acid esters. Complete dissolution is achieved within 30 min when powdered chitin is heated at 70 °C in a mixed solution of carboxylic acid(s) and trifluoroacetic anhydride. Chitin esters prepared are chitin acetate, chitin butyrate, chitin hexanoate and chitin octanoate, chitin co-acetate/butyrate, chitin co-acetate/hexanoate, chitin co-acetate/octanoate, chitin co-acetate/palmitate, each from a solution of the respective reactants. The products have degrees of O-acyl substitution in a range of DS 1-2 depending on the nature of acyl group, as analyzed by gas-liquid and high-pressure liquid chromatography. Acetic acid as a mutual component for the mixed-acid esters increases the total degree of substitution, and the acetyl substitution is close to the relative distribution in the reaction mixture for chitin co-acetate/butyrate. It is favored over hexanoate, octanoate, and palmitate. The parent molecules, as calculated by the composition of the chitin esters and their molecular weights by light-scattering spectroscopy, are 30 kDa for the smallest and 150-151 kDa for the largest. Films of these chitin derivatives when cast from solution are strong and flexible with limited extensibility. By dynamic mechanical analysis of the ester film, it was found that both the glass transition temperature (T_g) and the tensile modulus (E′ at 25 °C) are highest for chitin acetate (218 °C and 5.8 GPa), and lowest for chitin octanoate (182 °C and 1.5 GPa). For the other esters, these values lie between the above-cited values, where the T_g and the E′ decrease with an increase in the chain length of the acyl constituent.     

Preparation of fermentation-processed chitin and its application in chitinase affinity adsorption

A fermentation approach utilizing Paenibacillus sp. to process chitin was developed. The chitin obtained from this process is called fermentation-processed chitin (FPC), and it was further investigated with chitinase affinity adsorption studies together with three other adsorbents, i.e. crab shell chitin, colloid chitin, and enzyme-processed chitin. The results showed that FPC had the highest chitinase adsorption capacity. Under 15 °C and pH 5.0, FPC exhibited an optimal chitinase adsorption capacity of 85.9 U/g, which was 61.9% higher than that of the colloidal chitin. With 0.02 M acetic acid as the eluent, a purification-fold of 10.3 with 97% chitinase recovery was obtained. The results of surface morphology studies indicated that the FPC surface was modified to a fiber-like structure with deep pores. In comparison with the surface morphology of enzyme-processed chitin and colloidal chitin, it is inferred that the enhanced adsorption capacity of FPC for chitinase is attributed to both the effects of chitinase hydrolysis and the bacterial modification.     

Differentiations of Chitin Content and Surface Morphologies of Chitins Extracted from Male and Female Grasshopper Species

In this study, we used Fourier transform infrared spectroscopy (FT-IR), elemental analysis (EA), thermogravimetric analysis (TGA), X-ray diffractometry (XRD), and scanning electron microscopy (SEM) to investigate chitin structure isolated from both sexes of four grasshopper species. FT-IR, EA, XRD, and TGA showed that the chitin was in the alpha form. With respect to gender, two main differences were observed. First, we observed that the quantity of chitin was greater in males than in females and the dry weight of chitin between species ranged from 4.71% to 11.84%. Second, using SEM, we observed that the male chitin surface structure contained 25 – 90nm wide nanofibers and 90 – 250 nm nanopores, while no pores or nanofibers were observed in the chitin surface structure of the majority of females (nanofibers were observed only in M. desertus females). In contrast, the elemental analysis, thermal properties, and crystalline index values for chitin were similar in males and females. Also, we carried out enzymatic digestion of the isolated chitins using commercial chitinase from Streptomyces griseus. We observed that there were no big differences in digestion rate of the chitins from both sexes and commercial chitin. The digestion rates were for grasshoppers’ chitins; 88.45–95.48% and for commercial chitin; 94.95%.     

Chitin Amendment Increases Soil Suppressiveness toward Plant Pathogens and Modulates the Actinobacterial and Oxalobacteraceal Communities in an Experimental Agricultural Field

A long-term experiment on the effect of chitin addition to soil on the suppression of soilborne pathogens was set up and monitored for 8 years in an experimental field, Vredepeel, The Netherlands. Chitinous matter obtained from shrimps was added to soil top layers on two different occasions, and the suppressiveness of soil toward Verticillium dahliae, as well as plant-pathogenic nematodes, was assessed, in addition to analyses of the abundances and community structures of members of the soil microbiota. The data revealed that chitin amendment had raised the suppressiveness of soil, in particular toward Verticillium dahliae, 9 months after the (second) treatment, extending to 2 years following treatment. Moreover, major effects of the added chitin on the soil microbial communities were detected. First, shifts in both the abundances and structures of the chitin-treated soil microbial communities, both of total soil bacteria and fungi, were found. In addition, the abundances and structures of soil actinobacteria and the Oxalobacteraceae were affected by chitin. At the functional gene level, the abundance of specific (family-18 glycoside hydrolase) chitinase genes carried by the soil bacteria also revealed upshifts as a result of the added chitin. The effects of chitin noted for the Oxalobacteraceae were specifically related to significant upshifts in the abundances of the species Duganella violaceinigra and Massilia plicata. These effects of chitin persisted over the time of the experiment.     

2-Acylamido Analogues of N-Acetylglucosamine Prime Formation of Chitin Oligosaccharides by Yeast Chitin Synthase 2

Chitin, a homopolymer of β1,4-linked N-acetylglucosamine (GlcNAc) residues, is a key component of the cell walls of fungi and the exoskeletons of arthropods. Chitin synthases transfer GlcNAc from UDP-GlcNAc to preexisting chitin chains in reactions that are typically stimulated by free GlcNAc. The effect of GlcNAc was probed by using a yeast strain expressing a single chitin synthase, Chs2, by examining formation of chitin oligosaccharides (COs) and insoluble chitin, and by replacing GlcNAc with 2-acylamido analogues of GlcNAc. Synthesis of COs was strongly dependent on inclusion of GlcNAc in chitin synthase incubations, and N,N′-diacetylchitobiose (GlcNAc₂) was the major reaction product. Formation of both COs and insoluble chitin was also stimulated by GlcNAc₂ and by N-propanoyl-, N-butanoyl-, and N-glycolylglucosamine. MALDI analyses of the COs made in the presence of 2-acylamido analogues of GlcNAc showed they that contained a single GlcNAc analogue and one or more additional GlcNAc residues. These results indicate that Chs2 can use certain 2-acylamido analogues of GlcNAc, and likely free GlcNAc and GlcNAc₂ as well, as GlcNAc acceptors in a UDP-GlcNAc-dependent glycosyltransfer reaction. Further, formation of modified disaccharides indicates that CSs can transfer single GlcNAc residues.     

Direct Binding of a Plant LysM Receptor-like Kinase, LysM RLK1/CERK1, to Chitin in Vitro

Plants induce immune responses against fungal pathogens by recognition of chitin, which is a component of the fungal cell wall. Recent studies have revealed that LysM receptor-like kinase 1/chitin elicitor receptor kinase 1 (LysM RLK1/CERK1) is a critical component for the immune responses to chitin in Arabidopsis thaliana. However, the molecular mechanism of the chitin recognition by LysM RLK1 still remains unknown. Here, we present the first evidence for direct binding of LysM RLK1 to chitin. We expressed LysM RLK1 fused with yeast-enhanced green fluorescent protein (LysM RLK1-yEGFP) in yeast cells. Binding studies using the solubilized LysM RLK1-yEGFP and several insoluble polysaccharides having similar structures showed that LysM RLK1-yEGFP specifically binds to chitin. Subsequently, the fluorescence microscopic observation of the solubilized LysM RLK1-yEGFP binding to chitin beads revealed that the binding was saturable and had a high affinity, with a K_d of ∼82 nm. This binding was competed by the addition of soluble glycol chitin or high concentration of chitin oligosaccharides having 4–8 residues of N-acetyl glucosamine. However, the competition of these chitin oligosaccharides is weaker than that of glycol chitin. These data suggest that LysM RLK1 has a higher affinity for chitin having a longer residue of N-acetyl glucosamine. We also found that LysM RLK1-yEGFP was autophosphorylated in vitro and that chitin does not affect the phosphorylation of LysM RLK1-yEGFP. Our results provide a new dimension to chitin elicitor perception in plants.     

Isolation of Chitin-Specific Wheat Oxidoreductases

Anionic peroxidase (pI ～ 3.5) and oxalate oxidase (pI ～ 7.0) were isolated from wheat seedlings using chitin. The strength of binding of enzymes with chitin depended on the degree of its acetylation and the ionic strength of the buffer. It was assumed that the acetyl groups of chitin are involved in sorption of enzymes on this biopolymer. The ability of anionic peroxidase and oxalate oxidase for sorption on chitin allows this biopolymer to be used for isolation of these proteins from plants. Coadsorption of anionic peroxidase and oxalate oxidase on chitin suggests that these enzymes cooperate to ensure a defensive response of wheat against chitin-containing pathogens.     

A Metalloprotease (MprIII) Involved in the Chitinolytic System of a Marine Bacterium, Alteromonas sp. Strain O-7

Alteromonas sp. strain O-7 secretes several proteins in addition to chitinolytic enzymes in response to chitin induction. In this paper, we report that one of these proteins, designated MprIII, is a metalloprotease involved in the chitin degradation system of the strain. The gene encoding MprIII was cloned in Escherichia coli. The open reading frame of mprIII encoded a protein of 1,225 amino acids with a calculated molecular mass of 137,016 Da. Analysis of the deduced amino acid sequence of MprIII revealed that the enzyme consisted of four domains: the signal sequence, the N-terminal proregion, the protease region, and the C-terminal extension. The C-terminal extension (PkdDf) was characterized by four polycystic kidney disease domains and two domains of unknown function. Western and real-time quantitative PCR analyses demonstrated that mprIII was induced in the presence of insoluble polysaccharides, such as chitin and cellulose. Native MprIII was purified to homogeneity from the culture supernatant of Alteromonas sp. strain O-7 and characterized. The molecular mass of mature MprIII was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 115 kDa. The optimum pH and temperature of MprIII were 7.5 and 50°C, respectively, when gelatin was used as a substrate. Pretreatment of native chitin with MprIII significantly promoted chitinase activity. Furthermore, the combination of MprIII and a novel chitin-binding protease (AprIV) remarkably promoted the chitin hydrolysis efficiency of chitinase.     

Morphological changes induced by class III chitin synthase gene silencing could enhance penicillin production of Penicillium chrysogenum

Chitin synthases catalyze the formation of β-(1,4)-glycosidic bonds between N-acetylglucosamine residues to form the unbranched polysaccharide chitin, which is the major component of cell walls in most filamentous fungi. Several studies have shown that chitin synthases are structurally and functionally divergent and play crucial roles in the growth and morphogenesis of the genus Aspergillus although little research on this topic has been done in Penicillium chrysogenum. We used BLAST to find the genes encoding chitin synthases in P. chrysogenum related to chitin synthase genes in Aspergillus nidulans. Three homologous sequences coding for a class III chitin synthase CHS4 and two hypothetical proteins in P. chrysogenum were found. The gene which product showed the highest identity and encoded the class III chitin synthase CHS4 was studied in detail. To investigate the role of CHS4 in P. chrysogenum morphogenesis, we developed an RNA interference system to silence the class III chitin synthase gene chs4. After transformation, mutants exhibited a slow growth rate and shorter and more branched hyphae, which were distinct from those of the original strain. The results also showed that the conidiation efficiency of all transformants was reduced sharply and indicated that chs4 is essential in conidia development. The morphologies of all transformants and the original strain in penicillin production were investigated by light microscopy, which showed that changes in chs4 expression led to a completely different morphology during fermentation and eventually caused distinct penicillin yields, especially in the transformants PcRNAi1-17 and PcRNAi2-1 where penicillin production rose by 27 % and 41 %, respectively.     

Homogeneous synthesis and characterization of quaternized chitin in NaOH/urea aqueous solution

Water-soluble and white quaternized chitin (QC) was homogeneously synthesized by stirring transparent chitin solution (2%) in 8 wt%NaOH/4 wt% urea aqueous solution containing 2,3-Epoxypropyltrimethylammonium Chloride (EPTMAC) at 10 °C for 24 h. The structure and properties of quaternized chitin were characterized by FT-IR, XRD, ¹H NMR, GPC, element analysis and ζ-potential. The results indicate that quaternary groups were successfully incorporated onto chitin backbones and the degree of substitution (DS) of quaternary groups can be easily adjusted by changing the molar ratio of chitin unit to EPTMAC. Additionally, quaternized chitin shows better antibacterial activity against Escherichia coli and Staphylococcus aureus as compared with chitosan. Thus, this work provides a simply and “green” method to functionalize chitin and the resulting quaternized chitin may have potential applications in environmental, food and biomedical fields.     

Presence of LYM2 dependent but CERK1 independent disease resistance in Arabidopsis

Plants have the ability to detect invading fungi through the perception of chitin fragments released from the fungal cell walls. Plant chitin receptor consists of two types of plasma membrane proteins, CEBiP and CERK1. However, the contribution of these proteins to chitin signaling is different between Arabidopsis and rice. In Arabidopsis, it seems CERK1 receptor kinase is enough for both ligand perception and signaling, whereas both CEBiP and OsCERK1 are required for chitin signaling in rice. Here we report that Arabidopsis CEBiP homolog, LYM2, is not involved in chitin signaling but contributes to resistance against a fungal pathogen, Alternaria brassicicola, indicating the presence of a novel disease resistance mechanism in Arabidopsis.     

Chitin synthase genes in the arbuscular mycorrhizal fungus Glomus versiforme: full sequence of a gene encoding a class IV chitin synthase

Chitin synthase genes of the arbuscular mycorrhizal fungus Glomus versiforme were sought in an investigation of the molecular basis of fungal growth. Three DNA fragments (Gvchs1, Gvchs2 and Gvchs3) corresponding to the conserved regions of distinct chitin synthase (chs) genes were amplified by means of the polymerase chain reaction (PCR) with two sets of degenerate primers. Gvchs1 and Gvchs2 encode two class I chitin synthases, whereas Gvchs3 encodes a class IV chitin synthase. A genomic library was used to obtain the Gvchs3 complete gene (1194 amino acids), which shows a very close similarity to the class IV chitin synthase from Neurospora crassa.     

Fabrication and characterisation of α-chitin nanofibers and highly transparent chitin films by pulsed ultrasonication

α-Chitin nanofibers were fabricated with dried shrimp shells via a simple high-intensity ultrasonic treatment under neutral conditions (60 KHz, 300 W, pH = 7). The diameter of the obtained chitin nanofibers could be controlled within 20–200 nm by simply adjusting the ultrasonication time. The pulsed ultrasound disassembled natural chitin into high-aspect-ratio nanofibers with a uniform width (19.4 nm after 30 min sonication). The EDS, FTIR, and XRD characterisation results verified that α-chitin crystalline structure and molecular structure were maintained after the chemical purification and ultrasonic treatments. Interestingly, ultrasonication can slightly increase the degree of crystallinity of chitin (from 60.1 to 65.8). Furthermore, highly transparent chitin films (the transmittance was 90.2% at a 600 nm) and flexible ultralight chitin foams were prepared from chitin nanofiber hydrogels.     

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome.     

Effect of chitosan and other polyions on chitin deacetylase inRhizopus stolonifer

The effect of different concentrations of chitosan and other polyions on chitin deacetylase inRhizopus stolonifer was investigated. Crude protein extracts were analyzed for chitin deacetylase after native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Under native conditions, extracts fromRhizopus grown in the absence of chitosan exhibited one major and one minor acidic chitin deacetylase band, while extracts fromRhizopus grown in the presence of chitosan and two additional bands most clearly detected with treatment at 0.75 and 1.5 mg/ml. After SDS-PAGE, two major chitin deacetylase bands (at 40 and 110 kDa) and one very faint band (33 kDa) were present inRhizopus extracts. In the chitosan-grownRhizopus, the same bands appeared but were more intense and an additional band near 80 kDa was observed. The mycelial extracts ofRhizopus grown on potato dextrose broth amended with different polyions ord-glucosamine were also analyzed for chitin deacetylase. Chitosan,N,O-car☐ymethylchitosan and polyethylenimine increased the levels of chitin deacetylases inRhizopus, butd-glucosamine or polygalacturonate did not.