Showing papers on "Chitinase published in 1995"
TL;DR: This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains and decreases the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%.
Abstract: Three Enterobacter agglomerans strains which produce and excrete proteins with chitinolytic activity were found while screening soil-borne bacteria antagonistic to fungal plant pathogens. The chitinolytic activity was induced when the strains were grown in the presence of colloidal chitin as the sole carbon source. It was quantitated by using assays with chromogenic p-nitrophenyl analogs of disaccharide, trisaccharide, and tetrasaccharide derivatives of N-acetylglucosamine. A set of three fluorescent substrates with a 4-methylumbelliferyl group linked by (beta)-1,4 linkage to N-acetylglucosamine mono- or oligosaccharides were used to identify the chitinolytic activities of proteins which had been renatured following their separation by electrophoresis. This study provides the most complete evidence for the presence of a complex of chitinolytic enzymes in Enterobacter strains. Four enzymes were detected: two N-acetyl-(beta)-d-glucosaminidases of 89 and 67 kDa, an endochitinase with an apparent molecular mass of 59 kDa, and a chitobiosidase of 50 kDa. The biocontrol ability of the chitinolytic strains was demonstrated under greenhouse conditions. The bacteria decreased the incidence of disease caused by Rhizoctonia solani in cotton by 64 to 86%. Two Tn5 mutants of one of the isolates, which were deficient in chitinolytic activity, were unable to protect plants against the disease.
365 citations
TL;DR: Experiments with appropriate mutants showed that synthesis of the chitin combined with glucan is catalyzed by chit in synthetase 3, implying that chitInase can hydrolyze the linkage between N-acetylglucosamine and glucose.
336 citations
TL;DR: In this paper, a 1.1 kb rice genomic DNA fragment, containing a chitinase gene under the control of the CaMV 35S promoter, was cloned into the rice transformation vector pGL2.
Abstract: A 1.1 kb rice genomic DNA fragment, containing a chitinase gene under the control of the CaMV 35S promoter, was cloned into the rice transformation vector pGL2. After transformation of Indica rice protoplasts in the presence of polyethyleneglycol, plants were regenerated. The presence of the chimeric chitinase gene in T0 and T1 transgenic rice plants was detected by Southern blot analysis. Western blot analysis of transgenic plants and their progeny revealed the presence of two proteins with apparent molecular weights of 30 and 35 kDa that reacted with the chitinase antibody. Progeny from the chitinase-positive plants were tested for their resistance to the sheath blight pathogen, Rhizoctonia solani. The degree of resistance displayed by the transgenic plants to this pathogen correlated with the level of chitinase expression.
304 citations
TL;DR: Simultaneous expression of a tobacco class I chitinase and a class I β-1,3-glucanase gene in tomato resulted in increased fungal resistance, whereas transgenic tomato plants expressing either one of these genes were not protected against fungal infection.
Abstract: Simultaneous expression of a tobacco class I chitinase and a class I β-1,3-glucanase gene in tomato resulted in increased fungal resistance, whereas transgenic tomato plants expressing either one of these genes were not protected against fungal infection After infection with Fusarium oxysporum fsp lycopersici, a 36% to 58% reduction in disease severity was observed in resistant tomato lines Two transgenic lines largely recovered from the initial infection by the time wild-type tomato plants had died
301 citations
TL;DR: Among various divalent cations Mn2+ and Hg2+ completely inhibited the purified enzyme while beta-mercaptoethanol stimulated its activity, which had potential for cell wall lysis of many fungal pathogens tested.
Abstract: Streptomyces viridificans was found to be a good chitinase producer among nine species of Streptomyces screened. Minimum levels of constitutive enzyme were observed with both simple and complex carbon substrate. Arabinose doubled the enzyme production amongst the various pentoses and hexoses used with chitin. However, with glucose end-product inhibition and catabolite repression were observed. The enzyme tolerated a wide range of temperature (30-55 degrees C) and pH (3-7.5). Among various divalent cations Mn2+ and Hg2+ completely inhibited the purified enzyme while beta-mercaptoethanol stimulated its activity. Crude and purified enzyme had potential for cell wall lysis of many fungal pathogens tested.
229 citations
TL;DR: A hypothetical model for the binding of a hexasaccharide into the pronounced active site cleft of chitinase is built, consistent with a lysozyme-like mechanism of action in which Glu67 acts as proton donor and Glu89 is likely to stabilize the transition state oxycarbonium ion.
152 citations
TL;DR: Phylogenetic analyses indicate that AcMNPV, or an ancestral baculovirus, acquired the chitinase gene from a bacterium via horizontal gene transfer.
146 citations
TL;DR: Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1),PR-4, and PR-5.
Abstract: We studied the effect of constitutive expression of pathogenesis-related proteins (PRs) in tobacco plants on vesicular-arbuscular mycorrhiza. Tobacco lines genetically transformed to express various PRs constitutively under the control of the cauliflower mosaic virus 35S promoter of tobacco were examined. Immunoblot analysis and activity measurements demonstrated high levels of expression of the PRs in the root systems of the plants. Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1), PR-4, and PR-5. Similarly, constitutive expression of an acidic cucumber chitinase, of a basic tobacco chitinase with and without its vacuolar targeting peptide, of a basic (beta)-1,3-glucanase, and of combinations of PR-Q and PR-Q(prm1) or basic chitinase and basic (beta)-1,3-glucanase did not affect colonization by the mycorrhizal fungus. A delay of colonization by G. mosseae was observed in tobacco plants constitutively expressing the acidic isoform of tobacco PR-2, a protein with (beta)-1,3-glucanase activity.
126 citations
TL;DR: A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized and enzyme characteristics suggest a nutritional, rather than a morphogenetic, role for this chit inase.
Abstract: A gene (chit33) from the mycoparasitic fungus Trichoderma harzianum, coding for a chitinase of 33 kDa, has been isolated and characterized. Partial amino-acid sequences from the purified 33-kDa chitinase were obtained. The amino-terminal peptide sequence was employed to design an oligonucleotide probe and was used as a primer to isolate a 1.2-kb cDNA. The cDNA codes for a protein of 321 amino acids, which includes a putative signal peptide of 19 amino acids. All microsequenced peptides found in this sequence, indicate that this cDNA codes for the 33-kDa chitinase. A high homology (approximately 43% identity) was found with fungal and plant chitinases, including yeast chitinases. However enzyme characteristics suggest a nutritional (saprophytic or mycoparasitic), rather than a morphogenetic, role for this chitinase. The chit33 gene appears as a single copy in the T. harzianum genome, is strongly suppressed by glucose, and de-repressed under starvation conditions as well as in the presence of autoclaved mycelia and/or fungal cell walls. The 33-kDa chitinase seems to be very stable except under starvation conditions. The independent regulation of each of the chitinases in T. harzianum indicates different specific roles.
116 citations
TL;DR: Three chitinase inhibitors, styloguanidines 1, 2 and 3, were isolated from the sponge Stylotella aurantium collected from the Y ap sea as mentioned in this paper.
115 citations
TL;DR: Three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans are cloned and all three genes show striking similarity to other chitInase genes in the literature, especially in the proposed catalytic domain.
Abstract: Chitinase (EC 3.2.1.14) is an important enzyme for the remodeling of chitin in the cell wall of fungi. We have cloned three chitinase genes (CHT1, CHT2, and CHT3) from the dimorphic human pathogen Candida albicans. CHT2 and CHT3 have been sequenced in full and their primary structures have been analyzed: CHT2 encodes a protein of 583 aa with a predicted size of 60.8 kDa; CHT3 encodes a protein of 567 aa with a predicted size of 60 kDa. All three genes show striking similarity to other chitinase genes in the literature, especially in the proposed catalytic domain. Transcription of CHT2 and CHT3 was greater when C. albicans was grown in a yeast phase as compared to a mycelial phase. A transcript of CHT1 could not be detected in either growth condition.
TL;DR: Investigation ofStreptomyces olivaceoviridis as well as transformants of S. vinaceus and S. lividans carrying the exo-chiO1 gene on a multicopy vector secrete a 59-kDa chitinase which adheres strongly and under most conditions irreversibly to the substrate chit in, revealing a strong adhesion of the enzyme to its crystalline substrate via its binding domain is a prerequisite for efficient hydrolysis
Abstract: Streptomyces olivaceoviridis is an efficient chitin degrader. One of its genes encoding an exochitinase (exo-ChiO1) was previously characterized. The transcription was found to be inducible by chitin, but not by glucose. The transcriptional start site is situated 38 bp upstream of the start codon. S. olivaceoviridis as well as transformants of S. vinaceus and S. lividans carrying the exo-chiO1 gene on a multicopy vector secrete a 59-kDa chitinase which adheres strongly and under most conditions irreversibly to the substrate chitin. After having released the enzyme from the crystalline substrate in the presence of high concentrations of guanidine hydrochloride, it was purified to homogeneity by consecutive chitin- and immunoaffinity chromatographies. Immunofluorescence microscopy revealed that the enzyme specifically binds to crystalline alpha-chitin within fungi and other organisms as well as to beta-chitin, but not to colloidal chitin, chitosan, various types of cellulose, or other polysaccharides. The amino acids deduced from the highly specific binding domain (12 kDa) of this enzyme do not share significant similarity with any known region interacting with chitin or another substrate. During cultivation with chitin, the 59-kDa enzyme is proteolytically processed to a 47-kDa truncated chitinase lacking the chitin-binding domain. The 47-kDa enzyme hydrolyses crystalline chitin considerably less efficiently than the 59-kDa enzyme, whereas colloidal chitin and low-molecular-mass substrates are quite equally degraded by both enzymes at identical optimal pH (7.3) and temperature (45-55 degrees C) values. Thus a strong adhesion of the enzyme to its crystalline substrate via its binding domain is a prerequisite for efficient hydrolysis.
TL;DR: The hypothesis that insect chitinase has potential to enhance the insecticidal activity of entomopathogens is supported.
TL;DR: Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.
Abstract: Using colloidal [3H] chitin as a substrate, we provide the first demonstration of a chitinase in human leukocytes; chitinolytic activity in whole and disrupted leukocyte preparations (approximately 0.6 and 5.5 nmol of N-acetylglucosamine [GlcNAc] released min-1 mg of protein-1, respectively) was partially inhibited by the specific chitinase inhibitor allosamidin (9 microM). Following fractionation of the leukocytes, much higher levels of chitinase activity were detected in granulocyte-rich homogenates (approximately 7.2 nmol of GlcNAc released min-1 mg of protein-1) than in lymphocyte- and monocyte-rich homogenates (approximately 0.22 and 0.26 nmol of GlcNAc released min-1 mg of protein-1, respectively). Low levels of chitinase activity were detected in human serum (approximately 4 pmol of GlcNAc released min-1 mg of protein-1). Chitinolytic activity in granulocyte-rich homogenates and serum was partially inhibited by allosamidin (9 microM). Proteins with chitinolytic activities (approximate molecular masses, 48 and 56 kDa) distinct from lysozyme (14.3 kDa) were detected on polyacrylamide gels following the electrophoresis of human granulocyte-rich preparations. Chitinase activity, detected consistently in serum and leukocytes from all human volunteers investigated, may contribute to the protection of the host by cleaving chitin in the cell walls of fungal pathogens.
TL;DR: The data from this investigation suggest that the reduction of fungal development in induced plants results from at least two different mechanisms, one reduces penetration into the plant and the other restricts development of the fungus once it has penetrated.
TL;DR: To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid and salicylic acid is investigated.
Abstract: To study the possible involvement of plant hormones in the synthesis of stress proteins in tomato upon inoculation with Cladosporium fulvum, we investigated the induction of mRNAs encoding PR proteins and ethylene biosynthesis enzymes by ethephon, 2,6-dichloroisonicotinic acid (INA) and salicylic acid (SA) by northern blot analysis. Ethephon slightly induced some but not all mRNAs encoding intra- and extracellular PR proteins. INA induced all PR protein mRNAs analysed, except for intracellular chitinase and extracellular PR-4. SA induced all PR protein mRNAs analyzed, except for intracellular chitinase and osmotin. None of the inducers affected the expression of ACC synthase mRNA, whereas all three induced ethylene-forming enzyme (EFE) mRNA.
TL;DR: The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.
Abstract: SUMMARY: A gene encoding a chitinase from Serratia marcescens BJL200 was cloned and expressed in Escherichia coli and S. marcescens. Nucleotide sequencing revealed an open reading frame encoding a 55·5 kDa protein of 499 amino acids without a typical signal peptide for export. The cellular localization of the chitinase was studied, using two types of cell fractionation methods and immunocytochemical techniques. These analyses showed that the chitinase is located in the cytoplasm in E. coli, whereas it is exported to the periplasm in S. marcescens. Analysis of chitinase isolated from periplasmic fractions of S. marcescens carrying the cloned gene showed that export of the enzyme is not accompanied by processing at the N-terminus. The chitinase did not show any of the characteristics that have been proposed to direct the export of other non-processed extracellular proteins such as the E. coli haemolysin and might therefore be secreted via a hitherto unknown mechanism.
TL;DR: The data suggest that a signal produced during the first week after inoculation of leaf 1 affects protection and enzyme activities of leaf 2, the expanding leaves above leaf 2 and leaves which subsequently emerged from the bud in the absence of additional signal(s).
TL;DR: It is concluded that concanavalin B and family 18 chitinases are closely related, but that the protein has lost its enzymatic function and may act as a carbohydrate binding protein, however.
TL;DR: Assessment of the antifungal activity of crude protein preparations from such leaves, containing a complete set of the extra- and intracellular tomato chitinases and β-1,3-glucanases, by in vitro bioassays revealed that the test fungus Trichoderma viride is highly sensitive to the hydrolytic enzymes.
TL;DR: Radiometrie and gel electrophoretic analyses of crude extracts and extracellular washing fluids have shown that chitin deacetylase activity massively increases when the fungus starts to penetrate through the stomata, and that formation of the enzyme is strictly differentiation-specifically controlled.
Abstract: The broad bean rust fungus Uromyces viciae-fabae exhibits chitin only on surfaces of those infection structures which in nature are formed on the plant cuticle, but not on those differentiated in the intercellular space of the host leaf. Chitin deacetylase, an enzyme which converts chitin to chitosan, has been studied during in vitro differentiation of rust infection structures. Radiometrie and gel electrophoretic analyses of crude extracts and extracellular washing fluids have shown that chitin deacetylase activity massively increases when the fungus starts to penetrate through the stomata, and that formation of the enzyme is strictly differentiation-specifically controlled. The extracellular portion of chitin deacetylase activity was about 53% in 24-h-old differentiated infection structures. Five isoforms with apparent molecular masses of 48.1, 30.7, 25.2, 15.2 and 12.7 kDa were detectable after substrate SDS-PAGE. The enzyme is temperature-sensitive and has a pH optimum of 5.5-6.0.
TL;DR: The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chit inase A of Serratia marcescens.
Abstract: The chiA gene from Aeromonas caviae encodes an extracellular chitinase, 865 amino acids long, that shows a high degree of similarity to chitinase A of Serratia marcescens. Expression in Escherichia coli yielded an enzymatically active protein from which a leader sequence was removed, presumably during transport of the enzyme across the cell membrane.
TL;DR: This study suggests that species of zoo- and phyto-planktons, until now not reported to be associated with O1 and non-O1 V. cholerae, may act as reservoirs of these organisms as well as different species of Aeromonas in a fresh-water riverine ecosystem.
Abstract: Non-cultivable, pathogenic O1 and non-O1 Vibrio cholerae and Aeromonas spp. were resuscitated from aquatic arthropods and plant homogenate respectively, by rabbit ileal loop (RIL) assay. These organisms adhered to the aquatic arthropod Gerris spinolae and various species of phytoplankton in the River Ganga, but failed to grow after direct inoculation on artificial media except for only 10 homogenates of the arthropod. The number of non-O1 V. cholerae and Aeromonas recovered on direct inoculation of G. spinolae homogenates were in the order of 105–106 whereas those of the Ganga water were 102–103 ml−1. A total of 119 strains of O1 and non-O1 V. cholerae and Aeromonas spp. (69 isolates from G. spinolae and 50 from aquatic plants) were recovered from the loop contents. The results indicate that production of the enzyme chitinase by O1 and non-O1 V. cholerae and Aeromonas spp. might facilitate their adsorption and multiplication on different species of zoo- and phyto-plankton. Most of the isolates were enterotoxic, haemolytic and resistant to different antibiotics. This study suggests that species of zoo- and phyto-planktons, until now not reported to be associated with O1 and non-O1 V. cholerae, may act as reservoirs of these organisms as well as different species of Aeromonas in a fresh-water riverine ecosystem.
TL;DR: In this paper, the authors compared polymer-free reference system, the ATPS, and a polymer solution of PEG 20,000 and 5% dextran T500 aqueous two-phase systems for the production of extracellular chitinase from Serratia marcescens.
TL;DR: PAL and β-d-glucosidase variations appear to be suitable post-infectional markers of varietal resistance to P. capsici in pepper cell cultures but not in leaves.
TL;DR: Four ectomycorrhizal basidiomycetes and the oomycete Phytophthora cinnamomi were screened for chitinolytic activity in liquid culture and N -Acetylglucosaminidase, exo- and endo-chitinase activity were distinguished using fluorogenic 4-methylumbelliferyl substrates.
01 Jan 1995
TL;DR: In this article, the structure of concanavalin B has been determined at 1.65 A (1 A = 0.1 nm) resolution, where A is the catalytic glutamic acid.
Abstract: Seeds of Canavalia ensiformis (jack bean) contain besides large amounts of canavalin and concanavalin A, a protein with a molecular mass of 33,800 which has been named concanavalin B. Although concanavalin B shares about 40% sequence identity with plant chitinases belonging to glycosyl hydrolase family 18, no chitinase activity could be detected for this protein. To resolve this incongruity concanavalin B was crystallised and its three-dimensional structure determined at 1.65 A (1 A = 0.1 nm) resolution. The structure consists of a single domain with a (β/α)8 topology. A 30 amino acid residue long loop occurs between the second β-strand of the barrel and the second α-helix. This extended loop is unusual for the (β/α)8 topology, but appears in a similar conformation in the structures of the seed protein narbonin and several chitinases as well. Two non-proline cis-peptide bonds are present in the structure of concanavalin B: Ser34-Phe, and Trp265-Asn. This structural feature is rarely observed in proteins, but could also be identified in the three-dimensional structures of family 18 chitinases and narbonin in coincident positions. In the chitinases the aromatic residues of the non-proline cis-peptides have been proposed to have a function in the binding of the substrate. The region in concanavalin B, where in chitinases the active site is located, shows two significant differences. First, the catalytic glutamic acid is a glutamine in concanavalin B. Second, although part of the substrate binding cleft of the chitinases is present in concanavalin B, it is much shorter. From this we conclude that concanavalin B and family 18 chitinases are closely related, but that concanavalin B has lost its enzymatic function. It still may act as a carbohydrate binding protein, however.
TL;DR: It is proposed that CTS1 and CTS2 of Ci are members of two distinct classes of fungal chitinases, an observation not previously reported for a single fungus.
TL;DR: The isolated and sequenced a genomic clone corresponding to a class I basic endochitinase gene in pea, DAH53, which contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants.
Abstract: The fungicidal class I endochitinases (E.C.3.3.1.14, chitinase) are associated with the biochemical defense of plants against potential pathogens. We isolated and sequenced a genomic clone, DAH53, corresponding to a class I basic endochitinase gene in pea, Chil. The predicted amino acid sequence of this chitinase contains a hydrophobic C-terminal domain similar to the vacuole targeting sequences of class I chitinases isolated from other plants. The pea genome contains one gene corresponding to the chitinase DAH53 probe. Chitinase RNA accumulation was observed in pea pods within 2 to 4 h after inoculation with the incompatible fungal strain Fusarium solani f. sp. phaseoli, the compatible strain F. solani f.sp. pisi, or the elicitor chitosan. The RNA accumulation was high in the basal region (lower stem and root) of both fungus challenged and wounded pea seedlings. The sustained high levels of chitinase mRNA expression may contribute to later stages of pea's non-host resistance.
TL;DR: Six chitinases were purified from a culture supernatant of Aeromonas sp.
Abstract: Six chitinases were purified from a culture supernatant of Aeromonas sp. no. 10S-24 by ammonium sulfate precipitation, DEAE-Sephadex A-50, Butyl-Toyopearl 650M, and chromatofocusing. These enzymes were most active at pH 3.5–4.5 and the optimum temperature were 50°C. The molecular weights of the enzymes were 89,000 to 120,000 from SDS-polyacrylamide gel electrophoresis. N-Terminal amino acid sequences of the enzymes were similar to that of chitinase I.