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At least 307 records · Page 17Linked to original sources
PubMed2006

Improved Carica papaya tolerance to carmine spider mite by the expression of Manduca sexta chitinase transgene.

Papaya plants producing the tobacco hornworm (Manduca sexta) chitinase protein were obtained following microprojectile bombardment of embryogenic calli derived from the hypocotyls of the cultivar Kapoho. Polymerase chain reaction (PCR) was carried out to confirm the presence of the transgene. RT-PCR and a quantitative chitinase assay showed increased levels of chitinase activity in every selected transgenic line. Insect bioassays in the laboratory showed that plants expressing the Manduca sexta chitinase gene significantly inhibited multiplication of carmine spider mites (Tetranychus cinnabarinus Boisd.). Experiments conducted to evaluate reaction of the transgenic plants to natural infection by carmine spider mites showed that the Manduca sexta chitinase gene provided increased tolerance under field conditions.

Animals↗
PubMed1987

Chitin oligosaccharides as elicitors of chitinase activity in melon plants.

Chitin oligosaccharides elicited chitinase activity in melon plants. Hexamer to nonamer were the most efficient elicitors: hexamer for maximal stimulation of colorimetrically assessed chitinase activity, heptamer for maximal stimulation of radiochemically assessed chitinase activity. Chitinase elicitation was a rapid response to these elicitors: it occurred within 6 hours after treatment and was maximal at 12-24 hours. In addition, chitinase induction in melon plants by these oligosaccharides was both local and systemic.

Chitin↗
PubMed1993

Studies on chitosan: 6. Relationship between N-acetyl group distribution pattern and chitinase digestibility of partially N-acetylated chitosans.

The digestibility of partially N-acetylated chitosans by microbial chitinases was investigated in view of the distribution pattern of N-acetyl groups along the polysaccharide chain. Partially N-acetylated chitosans are classified into two groups; moderately N-deacetylated chitosans (MDC) with 10-30% acetyl content obtained by heterogeneous N-deacetylation of chitin and partially N-acetylated chitosans (PAC-H) with 20-70% acetyl content prepared by homogeneous N-acetylation of highly N-deacetylated chitosans (HDC). MDC have some blocks of N-acetyl-D-glucosamine (GlcNAc) sequences but PAC-H are random-type copolymers of GlcNAc and D-glucosamine. The apparent Km values of Streptomyces griseus chitinase were 0.14 g l-1 for 30% N-acetylated MDC and 0.16 g l-1 for 30% N-acetylated PAC-H. The Km values decreased with increased N-acetylation but the values for both MDC and PAC-H with similar acetyl content were almost the same. The chitinase from S. griseus could not distinguish the difference between block and random distributions of GlcNAc. The chitinases from Bacillus sp. and Bacillus sp. PI-7S also hydrolysed MDC and PAC-H in the same manner. From these results we conclude that sequences of GlcNAc are not necessary for recognition by these chitinases in contrast to lysozyme.

Acetylation↗
PubMed2005

Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum.

Protoplasts were isolated from Trichoderma harzianum strain PTh18 using lysing enzymes and self-fusion of T. harzianum protoplasts was carried out using polyethylene glycol in STC buffer. The fused protoplasts of T. harzianum were regenerated and 15 self-fusants were selected to study the chitinase production and biocontrol activity. High chitinase activity was measured in the culture filtrates of most of the self-fusants (87%) than the parent. Among the fusants, the strain SFTh8 produced maximum chitinase with a two-fold increase as compared to the parent strain. All the self-fusants exhibited increased antagonistic activity against Rhizoctonia solani than the parent. The crude chitinase preparation of SFTh8 lysed the mycelia of T. harzianum, Trichoderma viride and Trichoderma reesei and released the protoplasts in higher number than the crude chitinase preparation of parent strain PTh18.

Chitinases↗
PubMed2006

Cloning, purification, and characterization of chitinase from Bacillus sp. DAU101.

A chitinase encoding gene from Bacillus sp. DAU101 was cloned in Escherichia coli. The nucleotide sequencing revealed a single open reading frame containing 1781 bp and encoding 597 amino acids with 66 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram. The chitinase was composed of three domains: a catalytic domain, a fibronectin III domain, and a chitin binding domain. The chitinase was purified by GST-fusion purification system. The pH and temperature optima of the enzyme were 7.5 and 60 degrees C, respectively. The metal ions, Zn(2+), Cu(2+), and Hg(2+), were strongly inhibited chitinase activity. However, chitinase activity was increased 1.4-fold by Co(2+). Chisb could hydrolyze GlcNAc(2) to N-acetylglucosamine and was produced GlcNAc(2), when chitin derivatives were used as the substrate. This indicated that Chisb was a bifunctional enzyme, N-acetylglucosaminase and chitobiosidase. The enzyme could not hydrolyze glycol chitin, glycol chitosan, or CMC, but hydrolyzed colloidal chitin and soluble chitosan.

Amino Acid Sequence↗
PubMed2004

Molecular cloning of multiple chitinase genes in Japanese flounder, Paralichthys olivaceus.

Three different chitinase genes (fChi1, fChi2 and fChi3) were identified from Japanese flounder, Paralichthys olivaceus. The deduced amino-acid sequences of flounder chitinases revealed a typical chitinase structure containing a catalytic glyco-18 domain, a hinge region and a chitin binding domain type 2. The fChi1 and fChi2 mRNAs were predominantly expressed in the gastric glands of stomach. In contrast, expression of fChi3 was found in spleen, pancreas, stomach, intestine, liver, kidney and gonads of adult flounder by RT-PCR. The expression level of fChi3 in the adult tissues was below the detection limit of in situ hybridization (ISH) analysis; however, ISH signals were detected in the liver of flounder larvae. These results suggest that fChi1 and fChi2 are acidic chitinases that digest dietary chitin and that fChi3 probably is a macrophage specific chitinase (chitotriosidase) for biodefense and has an important unknown role in the liver during larval stages.

Amino Acid Sequence↗
PubMed2005

Methylxanthine drugs are chitinase inhibitors: investigation of inhibition and binding modes.

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 (K(i) of 37 microM). Crystallographic analysis of chitinase-inhibitor 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.

Chitinases↗
PubMed2006

Immunolocalization of a class III chitinase in two muskmelon cultivars reacting differently to Fusarium oxysporum f. sp. melonis.

Fusarium oxysporum f. sp. melonis is a highly specialized fungus that attacks the root system of melon (Cucumis melo L.). In this work the presence of a class III chitinase was examined by immunological techniques in the root and stem base of a susceptible (cv. Galia) and a resistant (cv. Bredor) melon during the infection process. By immunolocalization it was not possible to detect the constitutive presence of class III chitinase in any of the cultivars. However, the immunolabelling appeared in the root tissues of both cultivars as a consequence of wounding and of infection by F. oxysporum f. sp. melonis. Distinct patterns of chitinase detection were observed in the roots of the two cultivars as the infection progressed. Furthermore, by western blotting distinct class III chitinase isoforms were detected, which responded differently to the F. oxysporum f. sp. melonis infection. Our results strongly indicate that a relationship exists between class III chitinase and melon resistance to Fusarium infection, and that the resistance is associated with certain isoforms of this enzyme.

Chitinases↗
PubMed2006

Chitinases produced by Paenibacillus illinoisensis and Bacillus thuringiensis subsp. pakistani degrade Nod factor from Bradyrhizobium japonicum.

Chitinases are enzymes that hydrolyze internal beta-1,4-N-acetyl-D-glucosamine linkages of chitin. Since the backbone of Nod factors is a chitin oligomer, we investigated whether chitinases produced by soil bacteria Paenibacillus illinoisensis KJA-424 and Bacillus thuringiensis subsp. Pakistani HD 395 are able to degrade Nod factor produced by Bradyrhizobium japonicum, a phenomenon that could disrupt B. japonicum-soybean signaling and nodule establishment when chitinases are present. Purified Nod factor [LCO Nod Bj-V (C(18:1), MeFuc)] was isolated from Bradyrhizobium japonicum and incubated with crude chitinases isolated from KJA-424 and HD395, with or without acetate buffer. After 15 h of incubation, Nod factor in the resulting solution was quantified by HPLC. Degradation was greatest following treatment with KJA-424 (91.9%) and HD395 (86.5%) chitinases in acetate buffer. Treatments that included acetate buffer had higher levels of degradation than those without. For all treatments degradation was greater than 77%.

Acetates↗
PubMed2006

Cloning and overexpression of antifungal barley chitinase gene in Escherichia coli.

Plant chitinases are pathogenesis-related proteins, which are believed to be involved in plant defense responses to pathogen infection. In this study, chitinase gene from barley was cloned and overexpressed in Escherichia coli. Chitinase (35 kDa) was isolated and purified. Since the protein was produced as insoluble inclusion bodies, the protein was solubilized and refolded. Purified chitinase exerted broad-spectrum antifungal activity against Botrytis cinerea (blight of tobacco), Pestalotia theae (leaf spot of tea), Bipolaris oryzae (brown spot of rice), Alternaria sp. (grain discoloration of rice), Curvularia lunata (leaf spot of clover) and Rhizoctonia solani (sheath blight of rice). Due to the potential of broad-spectrum antifungal activity barley chitinase gene can be used to enhance fungal-resistance in crop plants such as rice, tobacco, tea and clover.

Antifungal Agents↗
PubMed2002

Quantitative production of 2-acetamido-2-deoxy-D-glucose from crystalline chitin by bacterial chitinase.

Finely powdered alpha- and beta-chitin can be completely hydrolyzed with chitinase (EC 3.2.1.14) and beta-N-acetylhexosaminidase (EC 3.2.1.52) for the production of 2-acetamido-2-deoxy-D-glucose (GlcNAc). Crude chitinase from Burkholderia cepacia TU09 and Bacillus licheniformis SK-1 were used to digest alpha- and beta-chitin powder. Chitinase from B. cepacia TU09 produced GlcNAc in greater than 85% yield from beta- and alpha-chitin within 1 and 7 days, respectively. B. licheniformis SK-1 chitinase completely hydrolyzed beta-chitin within 6 days, giving a final GlcNAc yield of 75%, along with 20% of chitobiose. However, only a 41% yield of GlcNAc was achieved from digesting alpha-chitin with B. licheniformis SK-1 chitinase.

Acetylglucosamine↗
PubMed2001

Enzyme activity determination on macromolecular substrates by isothermal titration calorimetry: application to mesophilic and psychrophilic chitinases.

Isothermal titration calorimetry has been applied to the determination of the kinetic parameters of chitinases (EC 3.2.1.14) by monitoring the heat released during the hydrolysis of chitin glycosidic bonds. Experiments were carried out using two different macromolecular substrates: a soluble polymer of N-acetylglucosamine and the insoluble chitin from crab shells. Different experimental temperatures were used in order to compare the thermodependence of the activity of two chitinases from the psychrophile Arthrobacter sp. TAD20 and of chitinase A from the mesophile Serratia marcescens. The method allowed to determine unequivocally the catalytic rate constant k(cat), the activation energy (E(a)) and the thermodynamic activation parameters (DeltaG(#), DeltaH(#), DeltaS(#)) of the chitinolytic reaction on the soluble substrate. The catalytic activity has also been determined on insoluble chitin, which displays an effect of substrate saturation by chitinases. On both substrates, the thermodependence of the activity of the psychrophilic chitinases was lower than that observed with the mesophilic counterpart.

Arthrobacter↗
PubMed1998

The Ld Cht1 gene encodes the secretory chitinase of the human pathogen Leishmania donovani.

Leishmania donovani promastigotes were shown to release chitinase activity during their growth in vitro. A PCR-based strategy identified a single copy ORF capable of encoding the L. donovani chitinase (Ld Cht1, 1374 bp). Ld Cht1 was shown to be actively transcribed by L. donovani promastigotes using reverse transcription and PCR amplification. The deduced aa sequence of Ld Cht1 showed high conservation to known chitinases including the putative active and two substrate binding sites. Antiserum generated against four peptides derived from its deduced aa sequence immunoprecipitated an approx. 50-kDa in vitro transcription/translation product of Ld Cht1. Further, this antiserum also immunoprecipitated both the native L. donovani 50-kDa Cht1 protein and the native chitinase activity synthesized and released by these parasites. Cumulatively, these data demonstrated that Ld Cht1 encoded the chitinase of this important human pathogen.

Amino Acid Sequence↗
PubMed2006

Cloning and expression of a chitinase gene from Sanguibacter sp. C4.

The chitinase Chi58 is an extracellular chitinase produced by Sanguibacter sp.strain C4. The gene-specific PCR primers were used to detect the presence of the chiA gene in strain C4. A chiA fragment (chiA-F) was amplified from the C4 genomic DNA and was used to blast-search the related sequences from the GenBank database. By alignment and selection of the highly conserved regions of the homologous sequences, two pairs of primers were designed to amplify the open reading frame (ORF) of the chitinase from strain C4 by nested PCR. The results revealed that the Chi58 ORF consisted of 1 692 nucleotides encoding a protein of 563 amino acid residues. The molecular weight of the mature protein was predicted to be 58.544 kDa. The Chi58 ORF was a modular enzyme composed of a signal peptide sequence, a polycystic kidney disease I domain, and a glycosyl hydrolase family 18 domain. The chitinase of C4 exhibited a high level of similarity to the chitinase A of Serratia (88.9%-99.6%) at the amino acid sequence level. The Chi58 gene was cloned into the expression vector pET32a to construct the recombinant plasmid pChi58 and was expressed in E. coli BL-21 (DE3) cells with IPTG induction. The molecular weight of the Trx-Chi58 fusion protein was estimated to be 81.1 kDa by SDS-PAGE.

Actinomycetales↗
PubMed2001

Antifungal activity of chitinases produced by some fluorescent pseudomonads against Colletotrichum falcatum Went causing red rot disease in sugarcane.

Chitinase production and growth of certain fluorescent pseudomonads isolated from sugarcane rhizosphere on different subtrates were studied. When chitin was substituted for glycerol in King's B medium, 3 of the 4 strains showed enhanced bacterial multiplication. Bacterial cells grown on chitin-containing medium showed enhanced antifungal activity against Colletotrichum falcatum Went causing red rot disease in sugarcane. Chitinase production was significantly higher when chitin was amended to King's B medium. Higher chitinase production was also recorded when fluorescent pseudomonad strains were grown in the medium containing crab-shell chitin. Cell-free bacterial culture filtrate from chitin-containing medium significantly inhibited mycelial growth of the pathogen. These cell-free conditioned media contained 3 to 7 polypeptides. Western blot analysis revealed five isoforms of chitinase with molecular masses of 47, 36, 32, 20 and 18.5 kDa. A possible role of chitinases in red rot disease management is discussed.

Antifungal Agents↗
PubMed2001

A genomic clone for a chitinase gene from the silkworm, Bombyx mori: structural organization identifies functional motifs.

A genomic clone containing the chitinase gene was isolated and characterized from the silkworm, Bombyx mori, using the polymerase chain reaction technique directed by primers designed from the chitinase cDNA of the insect previously cloned [Insect Biochem. Molec. Biol. 28 (1998) 163]. Results from nucleotide sequence analysis of the gene and its PCR amplification suggest that the B. mori genome probably has only one copy of chitinase gene. The gene is organized into 10 coding regions, exons, interrupted by noncoding regions, introns. The motifs encoded and exon organization of the gene are almost identical to the related gene from Manduca sexta. The results suggest that the domain organization of chitinase genes may be conserved among insect cuticular chitinase genes and that they are more complex than their counterparts in plants. The possibilities of intron splicing from the primary transcripts are also discussed.

Amino Acid Motifs↗
PubMed2003

Molecular characterization of Llchit1, a midgut chitinase cDNA from the leishmaniasis vector Lutzomyia longipalpis.

During development within the midgut of the sand fly vector, Leishmania parasites after undergoing differentiation and multiplication must escape the peritrophic matrix (PM). Although Leishmania chitinase is believed to take part in promoting the escape of the parasite from the PM by inducing degradation of chitin fibers, it is conceivable that a sand fly-derived chitinase can also have a role in such an event. Here we describe the molecular cloning and partial characterization of a complete cDNA from a putative gut-specific, blood-induced chitinase from the sand fly vector Lutzomyia longipalpis. Llchit1 has an ORF of 1425 bp that encodes a predicted 51.6 kDa mature protein showing high similarity with chitinases from several different organisms. Messenger RNA expression studies indicate that Llchit1 is detected only in the blood fed midgut and it seems to reach a peak at approximately 72 h post blood meal (PBM). To date, only one midgut-specific chitinase from an insect disease vector, AgChi-1 from Anopheles gambiae, has been characterized. As with its mosquito counterpart, Llchit1 can be a target for development of a transmission blocking vaccine.

Amino Acid Sequence↗
PubMed1997

Isolation and characterization of a genomic clone for the gene of an insect molting enzyme, chitinase.

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.

Amino Acid Sequence↗