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Direct detection of underivatized chitooligosaccharides produced through chitinase action using capillary zone electrophoresis

BLANES, Lucas; SAITO, Renata M.; GENTA, Fernando A.; DONEGA, Juliana; TERRA, Walter R.; FERREIRA, Clelia; LAGO, Claudimir Lucio do
Fonte: ACADEMIC PRESS INC ELSEVIER SCIENCE Publicador: ACADEMIC PRESS INC ELSEVIER SCIENCE
Tipo: Artigo de Revista Científica
ENG
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37%
Capillary electrophoresis with capacitively coupled contactless conductivity detection was successfully used to quantify N-acetylglucosamine and five N-acetyl-chitooligosaccharides (C2-C6) produced after reaction with a purified chitinase (TmChi) from Tenebrio molitor (Coleoptera). No derivatization process was necessary. The separation was developed using 10 mM NaOH with 10% (v/v) acetonitrile as background electrolyte and homemade equipment with a system that avoids the harmful effect of electrolysis. The limit of detection for all oligosaccharides was ca. 3 mu M, and the results indicated that the larger the oligosaccharide, the higher the sensitivity. Analysis of the chitooligosaccharides produced revealed that TmChi has an endolytic cleavage pattern with C5 as the best substrate (higher catalytic efficiency k(cat)/K-M) releasing C2 and C3. (c) 2007 Elsevier Inc. All rights reserved.

Disruption of the peritrophic matrix by exogenous chitinase feeding reduces fecundity in Lutzomyia longipalpis females

Araújo,Adriana Pereira Oliveira de; Telleria,Erich Loza; Dutra,Juliana da Matta Furniel; Júlio,Rute Maria; Traub-Csekö,Yara Maria
Fonte: Instituto Oswaldo Cruz, Ministério da Saúde Publicador: Instituto Oswaldo Cruz, Ministério da Saúde
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2012 EN
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Lutzomyia longipalpis is the most important vector of visceral leishmaniasis in Brazil. When female sandflies feed on blood, a peritrophic matrix (PM) is formed around the blood bolus. The PM is secreted by midgut cells and composed of proteins, glycoproteins and chitin microfibrils. The PM functions as both a physical barrier against pathogens present in the food bolus and blood meal digestion regulator. Previous studies of mosquitoes and sandflies have shown that the absence of a PM, resulting from adding an exogenous chitinase to the blood meal, accelerates digestion. In the present study, we analysed biological factors associated with the presence of a PM in L. longipalpis females. Insects fed blood containing chitinase (BCC) accelerated egg-laying relative to a control group fed blood without chitinase. However, in the BCC-fed insects, the number of females that died without laying eggs was higher and the number of eggs laid per female was lower. The eggs in both groups were viable and generated adults. Based on these data, we suggest that the absence of a PM accelerates nutrient acquisition, which results in premature egg production and oviposition; however, the absence of a PM reduces the total number of eggs laid per female. Reduced fecundity in the absence of a PM may be due to inefficient nutrient conversion or the loss of the protective role of the PM.

Cloning of the Bacillus thuringiensis serovar sotto chitinase (Schi) gene and characterization of its protein

Zhong,Wan-Fang; Fang,Ji-Chao; Cai,Ping-Zhong; Yan,Wen-Zhao; Wu,Jie; Guo,Hui-Fang
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2005 EN
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Chitinase plays a positive role in the pathogenicity of Bacillus thuringiensis to insect pests. We used touchdown PCR to clone the chitinase (Schi) gene from Bacillus thuringiensis serovar sotto (Bt sotto) chromosomal DNA. Our DNA sequencing analysis revealed that the Bt sotto Schi gene consists of an open reading frame (ORF) of 2067 nucleotides with codes for the chitinase precursor. We also found that the putative promoter consensus sequences (the -35 and -10 regions) of the Bt soto Schi gene are identical to those of the chiA71 gene from Bt Pakistani, the chiA74 gene from Bt kenyae and the ichi gene from Bt israelensis. The Schi chitinase precursor is 688 amino acids long with an estimated molecular mass of 75.75 kDa and a theoretical isoelectric point of 5.74, and contains four domains, which are, in sequence, a signal peptide, an N-terminal catalytic domain, a fibronectin type III like domain and a C-terminal chitin-binding domain. Sequence comparison and the evolutionary relationship of the Bt sotto Schi chitinase to other chitinase and chitinase-like proteins are also discussed.

Chitinase activities, scab resistance, mycorrhization rates and biomass of own-rooted and grafted transgenic apple

Schäfer,Tina; Hanke,Magda-Viola; Flachowsky,Henryk; König,Stephan; Peil,Andreas; Kaldorf,Michael; Polle,Andrea; Buscot,François
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/01/2012 EN
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This study investigated the impact of constitutively expressed Trichoderma atroviride genes encoding exochitinase nag70 or endochitinase ech42 in transgenic lines of the apple cultivar Pinova on the symbiosis with arbuscular mycorrhizal fungi (AMF). We compared the exo- and endochitinase activities of leaves and roots from non-transgenic Pinova and the transgenic lines T386 and T389. Local and systemic effects were examined using own-rooted trees and trees grafted onto rootstock M9. Scab susceptibility was also assessed in own-rooted and grafted trees. AMF root colonization was assessed microscopically in the roots of apple trees cultivated in pots with artificial substrate and inoculated with the AMF Glomus intraradices and Glomus mosseae. Own-rooted transgenic lines had significantly higher chitinase activities in their leaves and roots compared to non-transgenic Pinova. Both of the own-rooted transgenic lines showed significantly fewer symptoms of scab infection as well as significantly lower root colonization by AMF. Biomass production was significantly reduced in both own-rooted transgenic lines. Rootstock M9 influenced chitinase activities in the leaves of grafted scions. When grafted onto M9, the leaf chitinase activities of non-transgenic Pinova (M9/Pinova) and transgenic lines (M9/T386 and M9/T389) were not as different as when grown on their own roots. M9/T386 and M9/T389 were only temporarily less infected by scab than M9/Pinova. M9/T386 and M9/T389 did not differ significantly from M9/Pinova in their root chitinase activities...

Isolation and characterization of a chitinase gene from entomopathogenic fungus Verticillium lecanii

Zhu,Yanping; Pan,Jieru; Qiu,Junzhi; Guan,Xiong
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/06/2008 EN
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Entomopathogenic fungus Verticillium lecanii is a promising whitefly and aphid control agent. Chitinases secreted by this insect pathogen have considerable importance in the biological control of some insect pests. An endochitinase gene Vlchit1 from the fungus was cloned and overexpressed in Escherichia coli. The Vlchit1 gene not only contains an open reading frame (ORF) which encodes a protein of 423 amino acids (aa), but also is interrupted by three short introns. A homology modelling of Vlchit1 protein showed that the chitinase Vlchit1 has a (α/β)8 TIM barrel structure. Overexpression test and Enzymatic activity assay indicated that the Vlchit1 is a functional enzyme that can hydrolyze the chitin substrate, so the Vlchit1 gene can service as a useful gene source for genetic manipulation leading to strain improvement of entomopathogenic fungi or constructing new transgenic plants with resistance to various fungal and insects pests.

Molecular cloning of chitinase 33 (chit33) gene from Trichoderma atroviride

Matroudi,S.; Zamani,M.R.; Motallebi,M.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2008 EN
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In this study Trichoderma atroviride was selected as over producer of chitinase enzyme among 30 different isolates of Trichoderma sp. on the basis of chitinase specific activity. From this isolate the genomic and cDNA clones encoding chit33 have been isolated and sequenced. Comparison of genomic and cDNA sequences for defining gene structure indicates that this gene contains three short introns and also an open reading frame coding for a protein of 321 amino acids. The deduced amino acid sequence includes a 19 aa putative signal peptide. Homology between this sequence and other reported Trichoderma Chit33 proteins are discussed. The coding sequence of chit33 gene was cloned in pEt26b(+) expression vector and expressed in E. coli.

Production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans 191

Fleuri,Luciana F.; Kawaguti,Haroldo Y.; Sato,Hélia H.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2009 EN
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This study concerned the production, purification and application of extracellular chitinase from Cellulosimicrobium cellulans strain 191. In shaken flasks the maximum yield of chitinase was 6.9 U/mL after 72 h of cultivation at 25ºC and 200 rpm. In a 5 L fermenter with 1.5 vvm aeration, the highest yield obtained was 4.19 U/mL after 168 h of fermentation at 25ºC and 200 rpm, and using 3 vvm, it was 4.38 U/mL after 144 h of fermentation. The chitinase (61 KDa) was purified about 6.65 times by Sepharose CL 4B 200 gel filtration with a yield of 46.61%. The purified enzyme was able to lyse the cell walls of some fungi and to form protoplasts.

Chitinase Production by Streptomyces sp. ANU 6277

Narayana,Kolla J.P.; Vijayalakshmi,Muvva
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2009 EN
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Chitinase production by a terrestrial Streptomyces sp. ANU 6277 was studied under sub-merged fermentation. Chitinase production started after 24 h of incubation and reached maximum levels after 60 h of cultivation. A high level of chitinase activity was observed in the culture medium with pH 6 at 35ºC. Culture medium amended with 1% chitin was found to be suitable for maximum production of chitinase. An optimum concentration of colloidal chitin for chitinase production was determined. Studies on the influence of additional carbon and nitrogen sources on chitinase production revealed that starch and yeast extract served as good carbon and nitrogen sources to enhance chitinase yield.Chitinase was purified from crude enzyme extract by single step gel filtration by Sephadex G-100. Purified chitinase of the strain exhibited a distinct protein band near 45 kDa by means of SDS-PAGE.

Optimization of nutrition factors on chitinase production from a newly isolated Chitiolyticbacter meiyuanensis SYBC-H1

Hao,Zhikui; Cai,Yujie; Liao,Xiangru; Zhang,Xiaoli; Fang,Zhiyou; Zhang,Dabing
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/03/2012 EN
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The present study reports statistical medial optimization for chitinase production by a novel bacterial strain isolated from soil recently, which the name Chitinolyticbacter meiyuanensis SYBC-H1 is proposed. A sequential statistical methodology comprising of Plackett-Burman and response surface methodology (RSM) was applied to enhance the fermentative production of chitinase, in which inulin was firstly used as an effective carbon source. As a result, maximum chitinase activity of 5.17 U/mL was obtained in the optimized medium, which was 15.5-fold higher than that in the basal medium. The triplicate verification experiments were performed under the optimized nutrients levels which indicated that it well agreed with the predicted value.

Cloning and expression of the complement fixation antigen-chitinase of Coccidioides immitis.

Zimmermann, C R; Johnson, S M; Martens, G W; White, A G; Pappagianis, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1996 EN
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A chitinase had been isolated from the culture filtrates of Coccidioides immitis endosporulating spherules and from hyphae and shown to be the coccidioidal complement fixation (CF) and immunodiffusion-CF antigen. In the present study, we made use of our previously determined amino-terminal (N-terminal) sequence of the CF-chitinase to design degenerate oligonucleotide primers and to amplify and sequence a PCR product that coded for the N-terminal portion of the CF-chitinase. The PCR product was used as a hybridization probe to screen a developing spherule-(lambda)ZAP cDNA library, and three hybridizing clones were selected. These clones were converted into their pBluescript expression plasmid form in Escherichia coli and induced to express their recombinant proteins. Lysate from only one clone, pCTS 4-2A, yielded an enzymatically functional CF-chitinase and a line of identity with control immunodiffusion-CF-positive antigen. The pCTS 4-2A insert was sequenced and found to contain a deduced open reading frame coding for a 427-amino-acid polypeptide with an approximate molecular weight of 47 kDa. When purified by a chitin adsorption-desorption method, the recombinant protein exhibited virtually identical characteristics to those of the original C. immitis CF-chitinase. Nondenaturing gels of the pCTS 4-2A E. coli lysates and the purified C. immitis and recombinant CF-chitinase revealed proteins that had chitinase activity and similar relative electrophoretic mobilities. The appearance and relative levels of hybridizing RNA from the developing spherules-endospores (SEs) and hyphae correlated with the appearance or presence and level of CF-chitinase enzyme activity found in SEs culture filtrate and in cellular extracts of developing SE and hyphae. Thus...

Chitinase system of Bacillus circulans WL-12 and importance of chitinase A1 in chitin degradation.

Watanabe, T; Oyanagi, W; Suzuki, K; Tanaka, H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1990 EN
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Bacillus circulans WL-12, isolated as a yeast cell wall-lytic bacterium, secretes a variety of polysaccharide-degrading enzymes into culture medium. When chitinases of the bacterium were induced with chitin, six distinct chitinase molecules were detected in the culture supernatant. These chitinases (A1, A2, B1, B2, C, and D) showed the following distinct sizes and isoelectric points: Mr 74,000, pI 4.7 (A1); Mr 69,000, pI 4.5 (A2); Mr 38,000, pI 6.6 (B1); Mr 38,000, pI 5.9 (B2); Mr 39,000, pI 8.5 (C); and Mr 52,000, pI 5.2 (D). Among these chitinases, A1 and A2 had the highest colloidal-chitin-hydrolyzing activities. Chitinase A1 showed a strong affinity to insoluble substrate chitin. Purified chitinase A1 released predominantly chitobiose [(GlcNAc)2] and a trace amount of N-acetylglucosamine (GlcNAc) from colloidal chitin. N-terminal amino acid sequence analysis of chitinases A1 and A2 indicated that chitinase A2 was generated from chitinase A1, presumably by proteolytic removal of a C-terminal portion of chitinase A1. Since chitinase A2 did not have the ability to bind to chitin, the importance of the C-terminal region of chitinase A1 to the strong affinity of chitinase A1 to substrate chitin was suggested. Strong affinity of the chitinase seemed to be required for complete degradation of insoluble substrate chitin. From these results...

Cloning, characterization and expression of the chitinase gene of Enterobacter sp. NRG4

Salam, M.; Dahiya, N.; Sharma, R.; Soni, S. K.; Hoondal, G. S.; Tewari, R.
Fonte: Springer-Verlag Publicador: Springer-Verlag
Tipo: Artigo de Revista Científica
EN
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A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg−1. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the Km, kcat and catalytic efficiency (kcat/Km) values of recombinant chitinase were found to be 1.27 mg ml−1, 0.69 s−1 and 0.54 s−1M−1 respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.

Chitinase 3-Like 1 Promotes Macrophage Recruitment and Angiogenesis in Colorectal Cancer

Kawada, Mayumi; Seno, Hiroshi; Kanda, Keitaro; Nakanishi, Yuki; Akitake, Reiko; Komekado, Hideyuki; Kawada, Kenji; Sakai, Yoshiharu; Chiba, Tsutomu; Mizoguchi, Emiko
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN_US
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Chitinase 3-like 1 (CHI3L1), one of mammalian members of the chitinase family, is expressed in several types of human cancer, and elevated serum level of CHI3L1 is suggested to be a biomarker of poor prognosis in advanced cancer patients. However, the overall biological function of CHI3L1 in human cancers still remains unknown. Studies were performed to characterize the role of CHI3L1 in cancer pathophysiology utilizing human colorectal cancer samples and human cell lines. Plasma protein and tissue mRNA expression levels of CHI3L1 in colorectal cancer were strongly upregulated. Immunohistochemical analysis showed that CHI3L1 was expressed in cancer cells and CHI3L1 expression had a significant association with the number of infiltrated macrophages and microvessel density. By utilizing trans-well migration and tube formation assays, overexpression of CHI3L1 in SW480 cells (human colon cancer cells) enhanced the migration of THP-1 cells (human macrophage cells) and HUVECs (human endothelial cells), and the tube formation of HUVECs. The knockdown of CHI3L1 by RNA interference or the neutralization of CHI3L1 by anti-CHI3L1 antibody displayed strong suppression of CHI3L1-induced migration and tube formation. Cell proliferation assay showed that CHI3L1 overexpression significantly enhanced the proliferation of SW480 cells. ELISA analysis showed that CHI3L1 increased the secretion of inflammatory chemokines...

Overexpression, secretion and antifungal activity of the Saccharomyces cerevisiae chitinase

Carstens, M.; Vivier, M.; van Rensburg, P.; Pretorius, I.
Fonte: Inst Microbiologia Publicador: Inst Microbiologia
Tipo: Artigo de Revista Científica
Publicado em //2003 EN
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The excessive use of pesticides to prevent the outbreak of plant diseases holds various disadvantages for the cost-effectiveness of agricultural production practices, the quality of the end products, the environment and the labour force. The mounting public resistance to the use of hazardous pesticides on agricultural crops has hastened the search for natural antimicrobial peptides, enzymes and other types of biological control agents. The objective of this study was to investigate the potential of the antifungal activity of a yeast-derived chitinase for possible future agricultural applications. When plants are exposed to fungal pathogens, they produce pathogenesis-related (PR) defence proteins, such as chitinases, in order to degrade the chitin in the cell walls of the attacking fungi, thereby inhibiting further fungal growth and the development of hyphae. We have cloned the Saccharomyces cerevisiae chitinase gene (CTS1-2) into a multicopy 2μ-based plasmid and overexpressed it under the control of the phosphoglycerate kinase I gene (PGK1) promoter (PGK1P) and terminator (PGK1T) sequences. Secretion of the recombinant CTS1-2-encoded chitinases was directed by the native leader peptide, or by the secretion signals of either the yeast mating pheromone α-factor (MFα1S) or the Trichoderma reesei β-xylanase 2 (XYN2S). Northern blot analysis confirmed the PGK1PTdirected expression of CTS1-2 and an indirect enzyme assay was used to compare the efficiency of secretion of the three different recombinant chitinases. These recombinant chitinases were also shown to effectively inhibit spore germination and hyphal growth of Botrytis cinerea...

Over expression of rice chitinase gene in transgenic peanut (Arachis hypogaea L.) improves resistance against leaf spot

Iqbal, M.M.; Nazir, F.; Ali, S.; Asif, M.A.; Zafar, Y.; Iqbal, J.; Ali, G.M.
Fonte: Humana Press Publicador: Humana Press
Tipo: Artigo de Revista Científica
Publicado em //2012 EN
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A Rice chitinase-3 under enhance version of CaMV 35S was introduced into peanut (Arachis hypogaea L.) through Agrobacterium mediation. Agrobacterium tumefaciens strain LB4404 was used harboring the binary vector (pB1333-EN4-RCG3) containing the chitinase (chit) and hygromycin resistance (hpt) gene as selectable marker. Putative transgenic shoots were regenerated and grown on MS medium supplemented with 5 mg/l BAP, 1 mg/l kinetin, and 30 mg/l hygromycin. Elongated shoots were examined for the presence of the integrated rice chitinase gene along with hygromycin gene as selectable. The integration pattern of transgene in the nuclear genome of the putative transformed plants (T(0)) was confirmed through Southern hybridization analysis of the genomic DNA. Survival rate of the in vitro regenerated plantlets was over 60% while healthy putatively transgenic (T(0)) plants with over 42% transformation frequency were produced through Agrobacterium mediated gene transfer of the rice chitinase gene and all the plants flowered and set seed normally. T1 plants were tested for resistance against Cercospora arachidicola by infection with the microspores. Transgenic strains exhibited a higher resistance than the control (non-transgenic plants). chitinase gene expression in highly resistant transgenic strains was compared to that of a susceptible control. A good correlation was observed between chitinase activity and fungal pathogen resistance.; Muhammad Munir Iqbal...

Characterisation of the Role of LysM Receptor-Like Kinases and the CHIA Chitinase in the Perception of Peptidoglycan and in the Innate Immunity of Arabidopsis thaliana; Charakterisierung der Rolle der LysM Rezeptor-ähnlichen Kinasen und der CHIA Chitinase in der Perzeption von Peptidoglycan und der angeborenen Immunität von Arabidopsis thaliana

Grabherr, Heini Marjatta
Fonte: Universidade de Tubinga Publicador: Universidade de Tubinga
Tipo: Dissertação
EN
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Plants as sessile organisms cannot escape, when they are confronted with harmful pathogens. Instead, they are weaponed with sophisticated and highly complex molecular responses that allow them to defend themselves. The innate immunity forms the basis for the self-defense of higher organisms, including mammals, invertebrates and plants. During the basal immune response, conserved microbial signatures are perceived by pattern recognition receptors and trigger a variety of defense reactions leading to protection of the plant tissue and resistance. The bacterial cell wall component peptidoglycan (PGN) is one of such conserved signatures activating the plant defense responses, however the molecular mechanisms of its recognition was until now not understood. The importance of LysM-domain containing plant proteins in the recognition of carbohydrate ligands, such as chitin and lipochitooligosaccharides, has been elucidated in the last years. Due to the structural similarities between chitin, lipochitooligosaccharide and peptidoglycan, members of this protein family provide interesting candidates for a putative PGN receptor. The reverse genetics approach performed in this work revealed a LysM receptor kinase, CERK1, to be involved in PGN perception. CERK1 is not only essential for the PGN-mediated activation of defense responses in Arabidopsis thaliana...

Cloning and expression analysis of the chitinase gene Ifu-chit2 from Isaria fumosorosea

Meng,Huimin; Wang,Zhangxun; Meng,Xiangyun; Xie,Ling; Huang,Bo
Fonte: Sociedade Brasileira de Genética Publicador: Sociedade Brasileira de Genética
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2015 EN
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Entomopathogenic fungi can produce a series of chitinases, some of which function synergistically with proteases and other hydrolytic enzymes to degrade the insect cuticle. In the present study, the chitinase gene Ifu-chit2 from Isaria fumosorosea was investigated. The Ifu-chit2 gene is 1,435-bp long, interrupted by three short introns, and encodes a predicted protein of 423 amino acids with a 22 residue signal peptide. The predicted Ifu-Chit2 protein is highly homologous to Beauveria bassiana chitinase Bbchit2 and belongs to the glycohydrolase family 18. Ifu-Chit2 was expressed in Escherichia coli to verify chitinase activity, and the recombinant enzyme exhibited activity with a colloidal chitin substrate. Furthermore, the expression profiles of Ifu-chit2 were analyzed at different induction times under in vivo conditions. Quantitative real-time PCR analysis revealed that Ifu-chit2 expression peaked at two days post-induction. The expression of chitinase Ifu-chit2 in vivo suggests that the chitinase may play a role in the early stage of pathogenesis.

Expression and efficient secretion of a functional chitinase from Chromobacterium violaceum in Escherichia coli

Lobo, Marina Duarte Pinto; Silva, Fredy Davi Albuquerque; Landim, Patrícia Gadelha de Castro; Cruz, Paloma Ribeiro da; Brito, Thaís Lima de; Medeiros, Suelen Carneiro de; Oliveira, José Tadeu Abreu; Vasconcelos, Ilka Maria; Pereira, Humberto D'Muniz; G
Fonte: BioMed Central; London Publicador: BioMed Central; London
Tipo: Artigo de Revista Científica
ENG
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Background: Chromobacterium violaceum is a free-living β-proteobacterium found in tropical and subtropical regions. The genomic sequencing of C. violaceum ATCC 12472 has revealed many genes that underpin its adaptability to diverse ecosystems. Moreover, C. violaceum genes with potential applications in industry, medicine and agriculture have also been identified, such as those encoding chitinases. However, none of the chitinase genes of the ATCC 12472 strain have been subjected to experimental validation. Chitinases (EC 3.2.1.14) hydrolyze the β-(1,4) linkages in chitin, an abundant biopolymer found in arthropods, mollusks and fungi. These enzymes are of great biotechnological interest as potential biocontrol agents against pests and pathogens. This work aimed to experimentally validate one of the chitinases from C. violaceum. Results: The open reading frame (ORF) CV2935 of C. violaceum ATCC 12472 encodes a protein (439 residues) that is composed of a signal peptide, a chitin-binding domain, a linker region, and a C-terminal catalytic domain belonging to family 18 of the glycoside hydrolases. The ORF was amplified by PCR and cloned into the expression vector pET303/CT-His. High levels of chitinolytic activity were detected in the cell-free culture supernatant of E. coli BL21(DE3) cells harboring the recombinant plasmid and induced with IPTG. The secreted recombinant protein was purified by affinity chromatography on a chitin matrix and showed an apparent molecular mass of 43.8 kDa...

Characterization of a chitinase with antifungal activity from a native Serratia marcescens B4A

Zarei,Mandana; Aminzadeh,Saeed; Zolgharnein,Hossein; Safahieh,Alireza; Daliri,Morteza; Noghabi,Kambiz Akbari; Ghoroghi,Ahmad; Motallebi,Abbasali
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/09/2011 EN
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Chitinases have the ability of chitin digestion that constitutes a main compound of the cell wall in many of the phytopathogens such as fungi. In the following investigation, a novel chitinase with antifungal activity was characterized from a native Serratia marcescens B4A. Partially purified enzyme had an apparent molecular mass of 54 kDa. It indicated an optimum activity in pH 5 at 45ºC. Enzyme was stable in 55ºC for 20 min and at a pH range of 3-9 for 90 min at 25ºC. When the temperature was raised to 60ºC, it might affect the structure of enzymes lead to reduction of chitinase activity. Moreover, the Km and Vmax values for chitin were 8.3 mg/ml and 2.4 mmol/min, respectively. Additionally, the effect of some cations and chemical compounds were found to stimulate the chitinase activity. In addition, Iodoacetamide and Idoacetic acid did not inhibit enzyme activity, indicating that cysteine residues are not part of the catalytic site of chitinase. Finally, chitinase activity was further monitored by scanning electronic microscopy data in which progressive changes in chitin porosity appeared upon treatment with chitinase. This enzyme exhibited antifungal activity against Rhizoctonia solani, Bipolaris sp, Alternaria raphani, Alternaria brassicicola...

Comparison of isozyme transformation in maize as a result of insertion of the chitinase gene

Yan,PM; Zhang,HF; Wang,Q; Yan,XY; Sun,Y
Fonte: Phyton (Buenos Aires) Publicador: Phyton (Buenos Aires)
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2010 EN
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Isozymes of peroxidase (POD), catalase (CAT), esterase (EST) and superoxide dismutase (SOD) were analyzed on transgenic maize (with external chitinase gene) and its parent by vertical polyacrylamide gel electrophoresis (PAGE). This study was made using shoots at the fourth leaf stage. Results showed that: POD and EST were detected in 6 bands. POD-2 and POD-3 were present at the bud and seedling stages. POD-1, POD-4, POD-5 and POD-6 were only present at the seedling stage. POD-6 expressed stronger in the transgenic maize with chitinase than in its parent. EST-2 was present only at the bud stage, and its expression in transgenic maize was stronger than that in its parent. EST-5 only existed at the seedling stage. EST-4 did not exist in the parent maize seedlings and EST-1, EST-3 and EST-6 were present at the bud or seedling stage. Four bands were detected for CAT. CAT-1 and CAT-3 were weaker bands than the others. CAT-3 in transgenic maize was stronger than in its parent. Three bands of SOD were detected; SOD-1 and SOD-2 existed at the bud and seedling stages, but SOD-3 was not shown in buds of the parent corn. All data showed that the expression of isozymes in transgenic and parent maize had obvious differences.