Presence Of Chitinase Research Articles

PH/chitinase dual stimuli-responsive essential oil-delivery system based on mesoporous silica nanoparticles for control of rice blast.

Owing to their surface modifiability, smart mesoporous silica nanoparticles (MSNs) can be designed to respond to plant disease-microenvironmental stimuli, thereby achieving on-demand release of active ingredients to control disease by effectively improving citral (CT) stability. A pH/chitinase dual stimuli-responsive essential oil-delivery system (CT@HMS@CH/TA) was successfully fabricated by encapsulating CT in hollow mesoporous silica (HMS), and coating with tannic acid (TA) and chitosan (CH) within HMS by using the layer-by-layer assembly technique (LbL). CT@HMS@CH/TA with an average particle size of 125.12 ± 0.12 nm and a hollow mesoporous nanostructure showed high CT-loading efficiency (16.58% ± 0.17%). The photodegradation rate of CT@HMS@CH/TA under UV irradiation (48 h) was only 15.31%, indicating a 3.34-fold UV stability improvement. CT@HMS@CH/TA exhibited a higher CT release rate in response to acidic pH and the presence of chitinase, simulating the prevailing conditions as Magnaporthe oryzae infection. Furthermore, CT@HMS@CH/TA exhibited better adhesion without affecting normal rice growth, significantly upregulating chitinase gene expression and enhancing chitinase activity on M. oryzae, thus enhancing CT antifungal activity. CT@HMS@CH/TA improved CT stability and showed intelligent, controlled release-performance and higher antifungal efficacy, thus providing a new strategy for efficient application of essential oils for green control of rice blast disease. © 2024 Society of Chemical Industry.

Chemical and Molecular Composition of the Chrysalis Reveals Common Chitin-Rich Structural Framework for Monarchs and Swallowtails

The metamorphosis of a caterpillar into a butterfly is an awe-inspiring example of how extraordinary functions are made possible through specific chemistry in nature’s complex systems. The chrysalis exoskeleton is revealed and shed as a caterpillar transitions to butterfly form. We employed solid-state NMR to evaluate the chemical composition and types of biomolecules in the chrysalides from which Monarch and Swallowtail butterflies emerged. The chrysalis composition was remarkably similar between Monarch and Swallowtail. Chitin is the major polysaccharide component, present together with proteins and catechols or catechol-type linkages in each chrysalis. The high chitin content is comparable to the highest chitin-containing insect exoskeletons. Proteomics analyses indicated the presence of chitinases that could be involved in synthesis and remodeling of the chrysalis as well as cuticular proteins which play a role in the structural integrity of the chrysalis. The nearly identical 13C CPMAS NMR spectra of each chrysalis and similar structural proteins supports the presence of underlying design principles integrating chitin and protein partners to elaborate the chrysalis.

Protein content of the Hylesia metabus egg nest setae (Cramer [1775]) (Lepidoptera: Saturniidae) and its association with the parental investment for the reproductive success and lepidopterism

Hylesia metabus is a neotropical moth possessing toxic setae, which once in contact with the skin cause a severe dermatitis to humans known as lepidopterism. The only known function of the setae in the life cycle is to provide protection during the mating and egg-hatching stages. Approximately 65% of the protein content of the setae is a cluster of five proteases (28–45kDa) showing sequence homology to other S1A serine proteases. The N-glycans of a 40kDa protease are a mixture of neutral and sulfated G0F structures. The sulfated N-glycans have an important role in triggering the inflammatory response typical of lepidopterism while the proteolytic activity may promote the erosion of blood vessels and tissues causing focal hemorrhages. The presence of Chitinase and a 30kDa lipoprotein is probably related to the antifungal defense. In addition, chitin digestion of the setae may potentiate the inflammatory reaction caused by the toxins due to the formation of chitin adjuvants fragments. The combined effect of proteases and a chitinase may dissuade predating arthropods, by damaging their exoskeletons. Vitellogenin, a bacteriostatic protein, is able to recognize pathogen-associated patterns, which suggests its possible role in protecting the embryonated eggs from pathogenic microorganisms. SignificanceThe present study is the first report describing the different protein species present in the urticating egg nest setae of the neotropical moth Hylesia metabus - the most harmful of the Hylesia moths - causing a severe urticating dermatitis in humans known as lepidopterism. A distinctive feature of the venom is the presence of five different S1A serine proteases probably used to guarantee a more efficient degradation of a wider number of protein substrates. This work confirms that the presence of sulfated N-glycans is not an isolated finding since its presence has been demonstrated in two different proteases affirming that this PTM is of importance for the activation of the inflammatory response typical of lepidopterism. Additionally, this study gives useful information on the defense mechanisms used for protection of its progeny vs. vertebrate predators, fungus, bacteria or other arthropods such as ants. The proteins detected in the egg nest should be seen as an extended parental effort made by the females in order to achieve an optimal reproductive success, thus compensating for the considerable loss of progeny during the larval stages that seriously limits the number of sexually mature adults reaching the reproductive phase.

Chitin-degrading enzymes from an actinomycete ectosymbiont of Acromyrmex subterraneus brunneus (Hymenoptera: Formicidae)

Microbes have many mechanisms to exert their inhibitory activity against target pests. One such mechanism involves the production and secretion of hydrolytic enzymes, such as chitinases, which are produced naturally by plants in response to attack by insect herbivores and phytopathogens and have been sought as an additional factor to enhance pest management. Thus, our main aim was to screen the diverse actinomycete community associated with the integument of Acromyrmex subterraneus brunneus for a chitinase-producing strain and to characterize its chitinases. We identified isolate ENT-21—a Streptomyces sp.—as a chitinase-producer and our data indicate that this isolate produces a chitinolytic complex that contains a chitinase and a high-molecular-weight β-N-acetylglucosaminidase (>100 kDa) when cultured in Chitin-Czapek broth. The presence of chitinases in the genome of this isolate was checked by diagnostic PCR, and two chitinase genes belonging to family 18 group A and family 19 were verified. The chitinolytic activity of the crude extract was observed at pH values ranging from 3.8 to 11.0, with the highest chitinase activities recorded at pH 9.0 and 9.5, whereas optimum β-N-acetylglucosaminidase activity was observed over a narrow pH range, between pH 4.7 and 5.1. We describe some biochemical and molecular properties of the chitinase and β-N-acetylglucosaminidase produced by ENT-21, and discuss the potential for exploitation of these enzymes for pest control.

Analysis of a chitinase from EpapGV, a fast killing betabaculovirus

The main function of baculoviral chitinase protein (V-CHIA) is to promote the final liquefaction of infected host larvae, facilitating the dispersion of occlusion bodies (OBs) in the environment. In this study, a v-chiA from Epinotia aporema Granulovirus (EpapGV) was identified and characterized. The 1,713 base pairs long open reading frame encodes a protein of 570 amino acids with a predicted molecular weight of 63 kDa. EpapGV V-CHIA sequence alignment resulted 62 % identical to Pieris rapae GV and Blastp search revealed a high conservation among all baculovirus chitinases. Amino acid sequence analysis indicated that the C-terminal KDEL present in most alphabaculovirus chitinases is absent in EpapGV V-CHIA, as well as in the rest of the betabaculoviruses. Phylogenetic analysis was performed with bacterial, lepidopteran, and baculoviral chitinase sequences available in databases. Using an AcMNPV bacmid (bApGOZA) a recombinant Ac-chiAEpapGV was obtained in order to overexpress EpapGV V-CHIA in cell culture. The presence of chitinase was detected in purified AcMNPV-chiAEpapGV OBs. Peritrophic membranes of Anticarsia gemmatalis larvae fed with recombinant OBs showed an altered structure. The results presented in this study show that EpapGV chitinase overexpression in recombinant baculovirus can cause association of this protein with OBs, and suggest that this could be used to evaluate the protein role in early stages of baculoviral infections.

Bacterial Chitin Hydrolysis in Two Lakes with Contrasting Trophic Statuses

Chitin, which is a biopolymer of the amino sugar glucosamine (GlcN), is highly abundant in aquatic ecosystems, and its degradation is assigned a key role in the recycling of carbon and nitrogen. In order to study the significance of chitin decomposition in two temperate freshwater lakes with contrasting trophic and redox conditions, we measured the turnover rate of the chitin analog methylumbelliferyl-N,N'-diacetylchitobioside (MUF-DC) and the presence of chitinase (chiA) genes in zooplankton, water, and sediment samples. In contrast to the eutrophic and partially anoxic lake, chiA gene fragments were detectable throughout the oligotrophic water column and chiA copy numbers per ml of water were up to 15 times higher than in the eutrophic waters. For both lakes, the highest chiA abundance was found in the euphotic zone--the main habitat of zooplankton, but also the site of production of easily degradable algal chitin. The bulk of chitinase activity was measured in zooplankton samples and the sediments, where recalcitrant chitin is deposited. Both, chiA abundance and chitinase activity correlated well with organic carbon, nitrogen, and concentrations of particulate GlcN. Our findings show that chitin, although its overall contribution to the total organic carbon is small (~0.01 to 0.1%), constitutes an important microbial growth substrate in these temperate freshwater lakes, particularly where other easily degradable carbon sources are scarce.

BACILLUS THURINGIENSIS FERMENTATION OF WASTEWATER AND WASTEWATER SLUDGE – PRESENCE AND CHARACTERIZATION OF CHITINASES

This study investigated the presence of chitinases in Bacillus thuringiensis ssp kurstaki HD‐1(Bt) fermented broths of wastewater sludge (non‐hydrolyzed and hydrolyzed); starch industry wastewater and soyameal. Chitinase activity was absent in soyameal and present in others. Chitinase demonstrated peaks at pH 4.0 and temperatures 40 and 50°C with higher activity between pH 4–5 and 10–11. The chitinase band on SDS‐PAGE was found to be between 36 and 45 kDa for non‐hydrolyzed (NH) and hydrolyzed sludge (TH) and starch industry wastewater. The chitinase profile during fermentation showed peaks at 15 and 30 h for non‐hydrolyzed and hydrolyzed sludge and 15 and 24 h for starch industry wastewater. Chitinase retained 96–99 % activity after two weeks incubation at room temperature and pH 4. Bioassays with supplementation of Bt chitinases showed 1.2 fold increase in entomotoxicity of wastewater sludge and a small increase in starch industry wastewater. This study sheds light on production of Bt chitinases in alternative media which will have a long term effect on entomotoxicity of these formulations.

Evaluation of fungal culture filtrate containing chitinase as a biocontrol agent against Helicoverpa armigera

To evaluate the biocontrol efficacy of culture filtrate containing chitinase from Trichoderma harzianum against Heliothis. T. harzianum was cultured by submerged fermentation using colloidal chitin as sole carbon source. The ability of the culture filtrate to hydrolyse colloidal chitin indicated the presence of chitinase as one of its components. Biocontrol assay on Heliothis showed that the culture filtrate is a potent antifeedant as it reduced the feeding rate and body weight of the larvae. It reduced the successful pupation and increased larval and pupal mortality in a dosage-dependent manner when applied topically. The highest mortalities (70%) were recorded for groups treated with 2000 U ml(-1) chitinase activity. The percentage of adult emergence was zero for the highest chitinase concentration (2000 U ml(-1)) tried. The studies showed that the culture filtrate containing chitinase from T. harzianum is capable of negatively affecting the growth and metamorphosis of Heliothis larvae. In view of the need for safer and environmentally friendly pest management tools, the present study could help in the development of enzyme-based biopesticides against Heliothis.

Exploring the role of chitinolytic enzymes in the sea fan coral, Gorgonia ventalina

Chitinases are involved in defense against chitinaceous pathogens in both invertebrates and vertebrates. This study investigated whether sea fan corals, Gorgonia ventalina (Linnaeus) collected from the Florida Keys between the summer of 2002 and the summer of 2005 contain chitinases, and whether these enzymes could serve an analogous protective role against the fungal pathogen, Aspergillus sydowii (Thom et Church). Crude extracts of healthy sea fans contained detectible levels of exochitinase activity in an in vitro microplate assay using fluorogenic substrates. The exochitinase levels decreased upon injury, agitation, or manipulation of the tissue. A concurrent, transient increase of exochitinase in the surrounding water suggests that sea fans release chitinases as a response to these stresses. By contrast, endochitinase was detected in only 2 of 15 sea fans (13%), suggesting a high degree of variation for this enzyme. Sea fan chitinase-containing seawater and anion exchange chromatography fractions were both active against A. sydowii in an absorbance-based antifungal assay. The presence of chitinases in sea fan extracts, their release into the surrounding water upon stress, and their activity against A. sydowii suggests that further study of these enzymes in coral stress responses is warranted.

Chitosan cross-linking with a water-soluble, blocked diisocyanate. 2. Solvates and hydrogels.

A water-soluble, blocked diisocyante was used to cross-link chitosan under various degrees of solvation, including hydration to form hydrogels. Thermal cross-linking of films cast from various amounts of organic cosolvents was found to increase with increased level of cosolvent up to a solvation level of 17% (w/w) and to be more efficient than for films prepared without cosolvent. Rheological studies revealed that gel modulus increased and gel time decreased with increasing cross-linker content and that gelation kinetics were consistent with a process having an activation energy of 103 kJ/mol. Swelling of hydrogels indicated that, even at high levels of hydration, the increased molecular mobility of reactants allowed for efficient network formation in a concentration-dependent manner. The extent of solvation via equilibrium swelling correlated well with degradative properties of chitosan networks in the presence of Chitinase (E. C. 3.2.1.14) from Streptomyces griseus with stability increasing with decreasing swelling (i.e., increased cross-linking).

Presence of chitinase and beta-N-acetylglucosaminidase in the Aedes aegypti: a chitinolytic system involving peritrophic matrix formation and degradation

Measurement of the hydrolysis of specific fluorogenic substrates by spectrophotometry as well as the substrate activity–SDS–PAGE gel analysis of the chitinolytic activity in Aedes aegypti guts showed that both chitinase and β-N-acetylglucosaminidase are present and physiologically active. Both enzymes were present even in guts from unfed insects, but the activities increased rapidly after feeding on blood or an artificial protein-free diet. Chitinase activity was predominantly of the ‘endo’-type, reaching its maximum activity at 36 h and then declining to very low levels after the degradation of the peritrophic matrix (PM). Chitinase assay in gels after SDS–PAGE was a very sensitive method that allowed us to detect two chitinases with distinct molecular weights in the mosquito gut. Hydrolysis of a chitinase-specific substrate by chitinolytic activities in the mosquito guts was inhibited by allosamidin, a potent chitinase inhibitor. Allosamidin treatment led to the formation of an atypical thick PM, while the addition of exogenous chitinase completely blocked its formation. This chitinolytic system appears to operate both on the formation and degradation of the PM. Since the PM is involved in pathogen invasion, these results are important in facilitating a search for mechanisms that can block pathogen development in the mosquito vector.

Toxicity of Chitinase-Producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30°C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC50's of 4.93 × 104 and 1.32 × 105 spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC50's were reduced to 6.23 × 103 and 7.60 × 104 spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.

Involvement of chitinases of Bacillus thuringiensis during pathogenesis in insects.

Bacillus thuringiensis subsp. israelensis IPS78 and B. thuringiensis subsp. aizawai HD133 both secreted exochitinase activity when grown in a medium containing chitin. Allosamidin, a specific chitinase inhibitor, inhibited activity from both strains, with IC50 values of about 50 microM with colloidal chitin as substrate and between 1 and 10 microM with 4-methylumbelliferyl-diacetylchitobioside and 4-methylumbelliferyl-triacetylchitotrioside as substrates. The involvement of these chitinolytic activities during pathogenesis in insects has been investigated with B. thuringiensis subsp. israelensis IPS78 against larvae of the midge Culicoides nubeculosus, and with B. thuringiensis subsp. aizawai HD133 against caterpillars of the cotton leafworm Spodoptera littoralis. Presence of 100 microM allosamidin increased the LD50 by factors of 1.3 and 1.4, respectively, demonstrating a role for bacterial chitinases in the attack on the insects. Presence of chitinase A from Serratia marcescens considerably decreased the values for LD50 confirming previous observations with different systems of the potentiation of entomopathogenesis of B. thuringiensis by exogenous chitinases. The most likely action of the endogenous chitinases of B. thuringiensis is to weaken the insects' peritrophic membranes, allowing more ready access of the bacterial toxins to the gut epithelia. Addition of exogenous chitinases will then increase this effect. Complementary cross-infection experiments, strain HD133 against midge larvae and strain IPS78 against caterpillars, were performed to investigate the pathogen/host specificities of the effects. Results showed that much higher concentrations of bacteria were required to achieve even low mortalities, and addition of chitinase A gave no increase in death rate.

Autolysis in the anaerobic fungus piromyces communis OTS1 : Presence of Chitinase and β-1,3-glucanase activities

Autolysis in the anaerobic fungus piromyces communis OTS1 : Presence of Chitinase and β-1,3-glucanase activities

Nucellar callus of ?Femminello? lemon, selected for tolerance toPhoma tracheiphila toxin, shows enhanced release of chitinase and glucanase into the culture medium

Phoma tracheiphila is the causative agent of the disease mal secco. Citrus cultivars differ substantially in respect to their sensitivity to the pathogenP. tracheiphila and its toxin. Some cultivars (e.g., 'Femminello' lemon) are inherently sensitive while others (e.g., 'Tarocco' orange) are tolerant. Cell lines derived from nucellar tissue of 'Femminello', 'Tarocco' and a cell line selected for tolerance to the fungal toxin ('Femminello-S') were used to study host-pathogen interaction. Our results showed that calli or conditioned media of 'Tarocco' and 'Femminello-S' inhibited the size of co-cultivated fungal colonies when compared to 'Femminello'. In addition, conditioned medium of 'Tarocco' as well as 'FemminelloS', but not 'Femminello', promoted bursting of hyphal tips. A ten-fold increase in chitinase and glucanase enzymatic activity, as evaluated by radiometric assay and laminarin hydrolysis respectively, was detected in 'Femminello-S' extracellular extracts as compared to 'Femminello'. An increase in chitinase was also shown by immunoblot analysis. Our findings suggest a positive correlation between the presence of chitinase and glucanase in the conditioned media of the cultured cells and the tolerance of those cells toP. tracheiphila toxin.

Malaria parasite chitinase and penetration of the mosquito peritrophic membrane.

Malaria parasites (ookinetes) appear to digest the peritrophic membrane in the mosquito midgut during penetration. Previous studies demonstrated that lectins specific for N-acetylglucosamine bind to the peritrophic membrane and proposed that the membrane contains chitin [Rudin, W. & Hecker, H. (1989) Parasitol. Res. 75, 268-279]. In the present study, we show that the peritrophic membrane is digested by Serratia marcescens chitinase (EC 3.2.1.14), leading to the release of N-acetylglucosamine and fragmentation of the membrane. We also report the presence of a malaria parasite chitinase that digests 4-methylumbelliferyl chitotriose. The enzyme is not detectable until 15 hr after zygote formation, the time required for maturation of the parasite from a zygote to an ookinete, the invasive form of the parasite. At 20 hr, the enzyme begins to appear in the culture supernatant. The chitinase extracted from the parasite and found in the culture supernatant consists of a major band and two minor bands of activity on native polyacrylamide gel electrophoresis. The presence of chitin in the peritrophic membrane, the disruption of the peritrophic membrane during invasion, and the presence of chitinase in ookinetes suggest that the chitinase in ookinetes is used in the penetration of the peritrophic membrane.

Induction of chitinases and of translatable mRNA for these enzymes in melon plants infected with Collectotrichum lagenarium

In melon plants infected with Colletotrichum langenarium, there is a strong increase in the activity of chitinase, an enzyme with a potential defensive function against pathogens. In order to investigate the molecular mechanisms involved in the regulation of chitinase gene expression, antisera have been raised against two purified chitinases (I and II) from infected melon plants. Changes induced by infection in the rate of synthesis of chitinases were determined using direct immunoprecipitation of enzymes labelled in vivo with [ 35S]methionine. A large but transient increase in the rate of chitinase synthesis occurred 5 days after inoculation. The in vitro synthesis of chitinases was then studied in healthy and infected melon plants. Poly(A)RNA was fractionated by sucrose gradient density centrifugation, and translated in a rabbit reticulocyte lysate. The transition products were then separated by fast protein liquid chromatography (FPLC) and further analysed by SDS-polyacrylamide gel electrophoresis. The identification of in vitro synthesized chitinases was performed by immunoprecipitation. The obtained results indicated the presence of chitinases in in vitro translation products of mRNA from infected, but not from healthy melon seedlings. It is concluded therefore, that infection of melon seedlings by a pathogen caused an increase in the translatable mRNAs for host chitinases.

Presence and localization of chitinase in Hydra and Podocoryne (Cnidaria, Hydrozoa)

AbstractThe presence of chitinase was checked in three species of Hydrozoa by incubating γ‐chitin‐red (Hackmann and Goldberg, '64) with crude extracts of entire polyps or parts of them. When the substrate is lytically degraded it releases its soluble red dye the quantities of which were spectrophotometrically (λ = 510 nm) determined. Crude extracts of entire polyps of Hydra attenuata Pall., H. circumcincta M. Sch., and Podocoryne carnea M. Sars all contained active chitinase. A more detailed localization which was studied in H. attenuata revealed that the enzyme is confined to the endoderm of the body column and that it is absent in the ectoderm and in the tentacles which contain the stenotele nematocysts. Therefore, it is very unlikely that enzymatic action of chitinase aids the nematocyst in boring holes in the cuticule of the prey.

Chitinase activity in the crystalline style of the american oyster Crassostrea virginica

1. 1. Presence of chitinase in Crassostrea virginica crystalline styles was established. 2. 2. Specific activity of chitinase tended to increase towards the anterior portion of the style. 3. 3. Chitinase activity was not stimulated by chitin additions to 1 μm filtered sea water. 4. 4. Kaolin additions to the diet resulted in doubling chitinase specific activity.

The presence of chitinase in the digestive fluids of ants

Chitinolytic enzymes have been detected in the digestive fluids of seven species of ants, representing five subfamilies.