Showing papers on "Chitinase published in 2005"

Cloning of Beauveria bassiana chitinase gene Bbchit1 and its application to improve fungal strain virulence.

Abstract: Entomopathogenic fungi can produce a series of chitinases, some of which act synergistically with proteases to degrade insect cuticle. However, chitinase involvement in insect fungus pathogenesis has not been fully characterized. In this paper, an endochitinase, Bbchit1, was purified to homogeneity from liquid cultures of Beauveria bassiana grown in a medium containing colloidal chitin. Bbchit1 had a molecular mass of about 33 kDa and pI of 5.4. Based on the N-terminal amino acid sequence, the chitinase gene, Bbchit1, and its upstream regulatory sequence were cloned. Bbchit1 was intronless, and there was a single copy in B. bassiana. Its regulatory sequence contained putative CreA/Crel carbon catabolic repressor binding domains, which was consistent with glucose suppression of Bbchit1. At the amino acid level, Bbchit1 showed significant similarity to a Streptomyces avermitilis putative endochitinase, a Streptomyces coelicolor putative chitinase, and Trichoderma harzianum endochitinase Chit36Y. However, Bbchit1 had very low levels of identity to other chitinase genes previously isolated from entomopathogenic fungi, indicating that Bbchit1 was a novel chitinase gene from an insect-pathogenic fungus. A gpd-Bbchit1 construct, in which Bbchit1 was driven by the Aspergiullus nidulans constitutive promoter, was transformed into the genome of B. bassiana, and three transformants that overproduced Bbchit1 were obtained. Insect bioassays revealed that overproduction of Bbchit1 enhanced the virulence of B. bassiana for aphids, as indicated by significantly lower 50% lethal concentrations and 50% lethal times of the transformants compared to the values for the wild-type strain.

Chitinases and chitinase-like proteins in TH2 inflammation and asthma

Abstract: Chitin is the second most abundant biopolymer in nature, where it protects crustaceans, parasites, fungi, and other pathogens from the adverse effects of their environments, hosts, or both. Because chitin does not exist in mammals, it had been assumed that the chitinases that degrade it are also restricted to lower life forms. However, chitinases and chitinase-like proteins have recently been noted in mice and human subjects. The prototypic chitinase, acidic mammalian chitinase, was also noted to be induced during TH2 inflammation through an IL-13–dependent mechanism. It was also shown to play an important role in the pathogenesis of TH2 inflammation and IL-13 effector pathway activation and demonstrated to be expressed in an exaggerated fashion in human asthmatic tissues. The finding that chitinases contribute to host antiparasite responses and asthmatic TH2 inflammation support the concept that asthma might be a parasite-independent antiparasite response.

A complete survey of Trichoderma chitinases reveals three distinct subgroups of family 18 chitinases.

Abstract: Genome-wide analysis of chitinase genes in the Hypocrea jecorina (anamorph: Trichoderma reesei) genome database revealed the presence of 18 ORFs encoding putative chitinases, all of them belonging to glycoside hydrolase family 18. Eleven of these encode yet undescribed chitinases. A systematic nomenclature for the H. jecorina chitinases is proposed, which designates the chitinases corresponding to their glycoside hydrolase family and numbers the isoenzymes according to their pI from Chi18-1 to Chi18-18. Phylogenetic analysis of H. jecorina chitinases, and those from other filamentous fungi, including hypothetical proteins of annotated fungal genome databases, showed that the fungal chitinases can be divided into three groups: groups A and B (corresponding to class V and III chitinases, respectively) also contained the so Trichoderma chitinases identified to date, whereas a novel group C comprises high molecular weight chitinases that have a domain structure similar to Kluyveromyces lactis killer toxins. Five chitinase genes, representing members of groups A-C, were cloned from the mycoparasitic species H. atroviridis (anamorph: T. atroviride). Transcription of chi18-10 (belonging to group C) and chi18-13 (belonging to a novel clade in group B) was triggered upon growth on Rhizoctonia solani cell walls, and during plate confrontation tests with the plant pathogen R. solani. Therefore, group C and the novel clade in group B may contain chitinases of potential relevance for the biocontrol properties of Trichoderma.

Loss-of-function mutations in chitin responsive genes show increased susceptibility to the powdery mildew pathogen Erysiphe cichoracearum.

Abstract: Chitin is a major component of fungal walls and insect exoskeletons. Plants produce chitinases upon pathogen attack and chito-oligomers induce defense responses in plants, though the exact mechanism behind this response is unknown. Using the ATH1 Affymetrix microarrays consisting of about 23,000 genes, we examined the response of Arabidopsis (Arabidopsis thaliana) seedlings to chito-octamers and hydrolyzed chitin after 30 min of treatment. The expression patterns elicited by the chito-octamer and hydrolyzed chitin were similar. Microarray expression profiles for several genes were verified via northern analysis or quantitative reverse transcription-PCR. We characterized T-DNA insertion mutants for nine chito-oligomer responsive genes. Three of the mutants were more susceptible to the fungal pathogen, powdery mildew, than wild type as measured by conidiophore production. These three mutants included mutants of genes for two disease resistance-like proteins and a putative E3 ligase. The isolation of loss-of-function mutants with enhanced disease susceptibility provides direct evidence that the chito-octamer is an important oligosaccharide elicitor of plant defenses. Also, this study demonstrates the value of microarray data for identifying new components of uncharacterized signaling pathways.

Identification of an antifungal chitinase from a potential biocontrol agent, Bacillus cereus 28-9.

Abstract: Bacillus cereus 28-9 is a chitinolytic bacterium isolated from lily plant in Taiwan. This bacterium exhibited biocontrol potential on Botrytis leaf blight of lily as demonstrated by a detached leaf assay and dual culture assay. At least two chitinases (ChiCW and ChiCH) were excreted by B. cereus 28-9. The ChiCW-encoding gene was cloned and moderately expressed in Escherichia coli DH5α. Near homogenous ChiCW was obtained from the periplasmic fraction of E. coli cells harboring chiCW by a purification procedure. An in vitro assay showed that the purified ChiCW had inhibitory activity on conidial germination of Botrytis elliptica, a major fungal pathogen of lily leaf blight.

Enrichment of chitinolytic microorganisms: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain.

Abstract: Thirteen different chitin-degrading bacteria were isolated from soil and sediment samples. Five of these strains (SGE2, SGE4, SSL3, MG1, and MG3) exhibited antifungal activity against phytopathogenic fungi. Analyses of the 16S rRNA genes and the substrate spectra revealed that the isolates belong to the genera Bacillus or Streptomyces. The closest relatives were Bacillus chitinolyticus (SGE2, SGE4, and SSL3), B. ehimensis (MG1), and Streptomyces griseus (MG3). The chitinases present in the culture supernatants of the five isolates revealed optimal activity between 45°C and 50°C and at pH values of 4 (SSL3), 5 (SGE2 and MG1), 6 (SGE4), and 5–7 (MG3). The crude chitinase preparations of all five strains possessed antifungal activity. The chitinase of MG3 (ChiIS) was studied further, since the crude enzyme conferred strong growth suppression of all fungi tested and was very active over the entire pH range tested. The chiIS gene was cloned and the gene product was purified. The deduced protein consisted of 303 amino acids with a predicted molecular mass of 31,836 Da. Sequence analysis revealed that ChiIS of MG3 is similar to chitinases of Streptomyces species, which belong to family 19 of glycosyl hydrolases. Purified ChiIS showed remarkable antifungal activity and stability.

Signal Transduction by Tga3, a Novel G Protein α Subunit of Trichoderma atroviride

Abstract: Several fungi belonging to the genus Trichoderma can act as mycoparasites and are used commercially as biological control agents against plant-pathogenic fungi, such as Rhizoctonia solani, Botrytis cinerea, Sclerotium rolfsii, Sclerotinia sclerotiorum, and Pythium spp (5, 6, 18) Mycoparasitic strains can penetrate and kill the host fungi Mycoparasitism is accompanied by the secretion of antifungal metabolites, such as peptaibol antibiotics (32, 47), and morphological changes, such as coiling around the host and development of hooks and/or appressorium-like structures (11) Hydrolytic enzymes, such as chitinases, glucanases, and proteases, also are important for biocontrol activity, since they enable Trichoderma to degrade the host's cell wall and to utilize its cellular contents (20) Chitinase gene expression is induced by colloidal chitin, fungal cell walls, or the chitin monomer N-acetylglucosamine (29) In Trichoderma atroviride the N-acetylglucosaminidase (NAGase)-encoding gene, nag1, has a major impact on the induction by chitin of other chitinases (4) In mycoparasitic interactions between T atroviride and R solani, expression of ech42, which encodes endochitinase 42, is triggered by a low-molecular-weight diffusible factor released from the host prior to physical contact between the two fungi (10, 50) Lectins in the host's cell wall can induce coiling of the mycoparasite around the host (1, 24, 41) Elucidation of the signaling pathways underlying mycoparasitism has only recently begun Heterotrimeric G proteins are composed of α, β, and γ subunits Gα subunits play pivotal roles in the recognition process, virulence, and virulence-dependent development in a number of plant-pathogenic fungi (3, 15, 26, 27, 31) Fungal Gα subunits are highly conserved and can be divided into three major subgroups (3) In T atroviride, the subgroup I Gα protein Tga1 affects light-induced conidiation and mycoparasitism-related coiling (41) The corresponding protein in Trichoderma virens is involved in antagonism against S rolfsii but not in antagonism against R solani ΔtgaA loss-of-function mutants sporulate and have coiling behavior similar to that of the wild type (37), suggesting that the subgroup I Gα subunits have different functions in these mycoparasites The T virens subgroup II G protein α subunit, TgaB, has no specific role in development or mycoparasitism (37) Fungal subgroup III Gα proteins also regulate morphological and developmental processes, such as germination, conidiation, and secondary metabolite production (28, 53), and they may increase intracellular cyclic AMP (cAMP) levels by stimulating adenylyl cyclase (28, 33, 39) The objectives of this study were to isolate a subgroup III Gα subunit deletion mutant of T atroviride and to determine its role in development and mycoparasitism This report is the first report of the role of the Tga3-mediated G protein pathway in mycoparasitism-related properties, such as host recognition, chitinase expression, and secretion

Brief Communication Methylxanthine Drugs Are Chitinase Inhibitors: Investigation of Inhibition and Binding Modes

Abstract: Summary Family 18 chitinases play key roles in a range of pathogenic organisms and are overexpressed in the asthmatic lung. By screening a library of marketed drug molecules, we have identified methylxanthine derivatives as possible inhibitor leads. These derivatives, theophylline, caffeine, and pentoxifylline, are used therapeutically as antiinflammatory agents, with pleiotropic mechanisms of action. Here it is shown that they are also competitive inhibitors against a fungal family 18 chitinase, with pentoxifylline being the most potent (Ki of 37 M). Crystallographic analysis of chitinaseinhibitor complexes revealed specific interactions with the active site, mimicking the reaction intermediate analog, allosamidin. Mutagenesis identified the key active site residues, conserved in mammalian chitinases, which contribute to inhibitor affinity. Enzyme assays also revealed that these methylxanthines are active against human chitinases.

Novel insect resistance in Brassica napus developed by transformation of chitinase and scorpion toxin genes.

Abstract: Transgenic plants with introduced pest-resistant gene offer an efficient alternative insect control. The novel insect-resistant gene combination, chitinase(chi) and BmkIT(Bmk), containing an insect-specific chitinase gene and a scorpion insect toxin gene was introduced into Brassica napus cultivar via Agrobacterium-mediated transformation. Fifty-seven regenerated plantlets with kanamycin-resistance were obtained. Transgenic plants were verified by Southern blot analysis. Enzyme-linked immunosorbent assay (ELISA) and bioassay of artificial inoculation with diamondback moth (Plutella maculipenis) (DBM) larvae indicated that some of the transgenic plants were high-level expression for both chitinase and scorpion toxin proteins and performed high resistance against the tested pest infestation. The genetic analysis of T1 progeny confirmed that the inheritance of introduced genes followed the Mendelian rules.

Chitinase Genes in Lake Sediments of Ardley Island, Antarctica

Abstract: A sediment core spanning approximately 1,600 years was collected from a lake on Ardley Island, Antarctica. The sediment core had been greatly influenced by penguin guano. Using molecular methods, the chitinolytic bacterial community along the sediment core was studied over its entire length. Primers targeting conserved sequences of the catalytic domains of family 18 subgroup A chitinases detected group A chitinases from a wide taxonomic range of bacteria. Using quantitative competitive PCR (QC-PCR), chitinase gene copies in each 1-cm section of the whole sediment column were quantified. QC-PCR determination of the chitinase gene copies indicated significant correlation with phosphorus and total organic carbon concentration, suggesting a historical connection between chitinase gene copies and the amount of penguin guano input into the lake sediment. Most of the chitinase genes cloned from the historic sediment core were novel. Analysis of the chitinase gene diversity in selected sediment layers and in the fresh penguin deposits indicated frequent shifts in the chitinolytic bacterial community over time. Sequence analysis of the 16S rRNA genes of chitinolytic bacteria isolated from the lake sediment revealed that the isolates belonged to Janthinobacterium species, Stenotrophomonas species of γ-Proteobacteria, Cytophaga species of the Cytophaga-Flexibacter-Bacteroides group, and Streptomyces and Norcardiopsis species of Actinobacteria. Chitinase gene fragments were cloned and sequenced from these cultivated chitinolytic bacteria. The phylogeny of the chitinase genes obtained from the isolates did not correspond well to that of the isolates, suggesting acquisition via horizontal gene transfer.

Purification and characterization of an extracellular chitinase from the antifungal biocontrol agent Streptomyces halstedii

Abstract: An extracellular chitinase from Streptomyces halstedii AJ-7, a broad spectrum antifungal biocontrol agent, was characterized and purified. The apparent molecular weight of the purified protein was 55 kDa, Km value and Vmax of the protein for colloidal chitin were 3.2 mg ml−1 and 118 μmol h−1, respectively. The growth and chitinase activity of S. halstedii AJ−7 were enhanced by adding of 0.1% killed mycelium of Fusarium oxysporium in a medium containing 0.2% colloidal chitin.

Purification and characterization of a thermostable chitinase from Bacillus licheniformis Mb-2

Abstract: A chitinase produced by Bacillus licheniformis MB-2 isolated from Tompaso geothermal springs, Indonesia, was purified and characterized. The extracellular enzyme was isolated by successive hydrophobic interaction, anion exchange, and gel filtration chromatographies. The purified enzyme was a monomer with an apparent molecular weight of 67 kDa. The optimal temperature and pH of the enzyme were 70 °C and 6.0, respectively. It was stable below 60 °C for 2 h and over a broad pH range of 4.0–11.0 for 4 h. The enzyme was resistant to denaturation by urea (1 M), Tween-20 (1%) and Triton-X (1%), but unstable toward organic solvents such as dimethyl sulphoxide, DMSO, (5%) and polyethylene glycol, PEG, (5%) for 30 min. The enzyme hydrolysed colloidal chitin, glycol chitin, chitosan, and glycol chitosan. The first 13 N-terminal amino acids of the enzyme were determined as SGKNYKIIGYYPS, which is identical to those in chitinases from B. licheniformis and B. circulans.

Detection of pathogenesis-related proteins-chitinase and β-1,3-glucanase in induced chickpea

Abstract: Pathogenesis-related (PR) proteins, chitinase and β-1,3-glucanase were extracted from induced chickpea plant and purified by gel filtration. Time-course accumulation of these PR-proteins in induced chickpea plants was significantly (P = 0.05) higher than the control. Maximum activities of these PR-proteins were recorded after three days of inoculation in all induced plants. Thereafter, the activity decreased progressively. Two chitinases and three β-1,3-glucanases were detected in induced chickpea plants. The molecular mass of the purified chitinases was 31 and 62 kDa and β-1,3-glucanases was 23, 27 and 39 kDa. Purified chitinases and β-1,3-glucanases also inhibited growth of Fusarium oxysporum f. sp. ciceri and other phytopathogenic fungi.

Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki

Abstract: Aims: The present work aims to study a new chitinase from Bacillus thuringiensis subsp. kurstaki. Methods and Results: BUPM255 is a chitinase-producing strain of B. thuringiensis, characterized by its high chitinolytic and antifungal activities. The cloning and sequencing of the corresponding gene named chi255 showed an open reading frame of 2031 bp, encoding a 676 amino acid residue protein. Both nucleotide and amino acid sequences similarity analyses revealed that the chi255 is a new chitinase gene, presenting several differences from the published chi genes of B. thuringiensis. The identification of chitin hydrolysis products resulting from the activity, exhibited by Chi255 through heterologous expression in Escherichia coli revealed that this enzyme is a chitobiosidase. Conclusions: Another chitinase named Chi255 belonging to chitobiosidase class was evidenced in B. thuringiensis subsp. kurstaki and was shown to present several differences in its amino acid sequence with those of published ones. The functionality of Chi255 was proved by the heterologous expression of chi255 in E. coli. Significance and Impact of the Study: The addition of the sequence of chi255 to the few sequenced B. thuringiensis chi genes might contribute to a better investigation of the chitinase ‘structure-function’ relation.

Production of an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

Abstract: In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.

Characterization and antifungal activity of gazyumaru (Ficus microcarpa) latex chitinases: both the chitin-binding and the antifungal activities of class I chitinase are reinforced with increasing ionic strength.

Abstract: Three chitinases, designated gazyumaru latex chitinase (GLx Chi)-A, -B, and -C, were purified from the latex of gazyumaru (Ficus microcarpa). GLx Chi-A,-B, and -C are an acidic class III (33 kDa, pI 4.0), a basic class I (32 kDa, pI 9.3), and a basic class II chitinase (27 kDa, pI > 10) respectively. GLx Chi-A did not exhibit any antifungal activity. At low ionic strength, GLx Chi-C exhibited strong antifungal activity, to a similar extent as GLx Chi-B. The antifungal activity of GLx Chi-C became weaker with increasing ionic strength, whereas that of GLx Chi-B became slightly stronger. GLx Chi-B and -C bound to the fungal cell-walls at low ionic strength, and then GLx Chi-C was dissociated from them by an escalation of ionic strength, but this was not the case for GLx Chi-B. The chitin-binding activity of GLx Chi-B was enhanced by increasing ionic strength. These results suggest that the chitin-binding domain of basic class I chitinase binds to the chitin in fungal cell walls by hydrophobic interaction and assists the antifungal action of the chitinase.

Production of N -acetyl-β- d -glucosamine from chitin by Aeromonas sp. GJ-18 crude enzyme

Abstract: A bacterium, GJ-18, having strong chitinolytic activity was isolated from coastal soil. The isolated strain was identified as Aeromonas sp. by morphological and biochemical properties along with 16S rRNA gene sequence. The crude chitinolytic activity of culture supernatants was maximal on the 5th day of culture. Below 45°C, chitin was effectively hydrolyzed to N-acetyl-β-d-glucosamine (GlcNAc) by Aeromonas sp. GJ-18 crude enzymes, but hydrolysis decreased markedly above 50°C. The optimum pH for enzyme activity was 5.0. TLC and HPLC analysis revealed that, below 45°C, the major reaction product was GlcNAc with a small amount of (GlcNAc)2 and (GlcNAc)3, whereas above 50°C the major product was (GlcNAc)2. When swollen chitin (100 mg) was incubated with crude enzyme preparations (10 U) at 40°C, chitin was hydrolyzed to 83.0 and 94.9% yield of GlcNAc within 5 and 9 days, respectively.

Purification, characterization, and antifungal activity of chitinases from pineapple (Ananas comosus) leaf.

Abstract: Three chitinases, designated pineapple leaf chitinase (PL Chi)-A, -B, and -C were purified from the leaves of pineapple (Ananas comosus) using chitin affinity column chromatography followed by several column chromatographies. PL Chi-A is a class III chitinase having a molecular mass of 25 kDa and an isoelectric point of 4.4. PL Chi-B and -C are class I chitinases having molecular masses of 33 kDa and 39 kDa and isoelectric points of 7.9 and 4.6 respectively. PL Chi-C is a glycoprotein and the others are simple proteins. The optimum pHs of PL Chi-A, -B, and -C toward glycolchitin are pH 3, 4, and 9 respectively. The chitin-binding ability of PL Chi-C is higher than that of PL Chi-B, and PL Chi-A has lower chitin-binding ability than the others. At low ionic strength, PL Chi-B exhibits strong antifungal activity toward Trichoderma viride but the others do not. At high ionic strength, PL Chi-B and -C exhibit strong and weak antifungal activity respectively. PL Chi-A does not have antifungal activity.

Roles of four chitinases (chia, chib, chic, and chid) in the chitin degradation system of marine bacterium Alteromonas sp. strain O-7

Abstract: Alteromonas sp. strain O-7 secretes four chitinases (ChiA, ChiB, ChiC, and ChiD) in the presence of chitin. To elucidate why the strain produces multiple chitinases, we studied the expression levels of the four genes and proteins, their enzymatic properties, and their synergistic effects on chitin degradation. Among the four chitinases, ChiA was produced in the largest quantities, followed by ChiD, and the production of ChiB and ChiC changed at lower levels than those of ChiA and ChiD. The expression of the chiA, chiB, chiC, and chiD genes was investigated at the transcriptional level. The RNA transcript of chiA was most strongly induced in the presence of chitin, the expression of chiD followed, and the RNA transcripts of chiB and chiC changed at low levels. The hydrolyzing activities of the four chitinases against various substrates were examined. ChiA was the most active enzyme against powdered chitin, whereas ChiC was the most active against soluble chitin among the four chitinases. ChiD had activities closer to those of ChiA than to those of ChiB and ChiC. ChiB showed no distinctive feature against the chitinous substrates tested. When powdered chitin was treated with the proper combination of four chitinases, an approximately 2.0-fold increase in the hydrolytic activity was observed. These results, together with the results described above, indicate that ChiA plays a central role in chitin degradation for this strain.

Boophilus microplus Infection by Beauveria amorpha and Beauveria bassiana : SEM Analysis and Regulation of Subtilisin-like Proteases and Chitinases

Abstract: Beauveria bassiana is a well-known broad-range arthropod pathogen which has been used in biological control of several pest insects and ticks such as Boophilus microplus. Beauveria amorpha has both endophytic and entomopathogenic characteristics, but its capacity for biological control has still not been studied. During the processes of host infection, B. bassiana and B. amorpha produce several hydrolytic extracellular enzymes, including proteases and chitinases, which probably degrade the host cuticle and are suggested to be pathogenicity determinants. To access the role of these enzymes during infection in the tick B. microplus, we analyzed their secretion during fungus growth in single and combined carbon sources, compared to complex substrates such as chitin and B. microplus cuticle. Chitin and tick cuticle-induced chitinase in both fungus and protease was induced only by tick cuticle. SEM analysis of B. amorpha and B. bassiana infecting B. microplus showed apressorium formation during penetration on cattle tick cuticle.