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1.
Food Chem ; 305: 125483, 2020 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-31610420

RESUMO

Kiwifruit (Actinidia deliciosa cv. Jinkui) were treated with 0.1 mmol/L methyl jasmonate (MeJA) to investigate the effects on disease resistance to soft rot caused by Botryosphaeria dothidea. The results showed that MeJA treatment significantly reduced the diameter of lesions after inoculation with B. dothidea. This treatment significantly enhanced the activities of related antioxidant protective enzymes, defence-related enzymes including catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), polyphenol oxidase (PPO), chitinase (CHI), ß-1,3 glucanase (GLU) and increased the accumulation of total phenolic content, while the degree of membrane lipid peroxidation was reduced. MeJA treatment effectively enhanced gene expression of AcPOD, AcSOD, AcCHI and AcGLU. The results from this research suggest that MeJA treatment is a promising and safe strategy for controlling postharvest rot soft of kiwifruit.


Assuntos
Acetatos/farmacologia , Actinidia/microbiologia , Ascomicetos/efeitos dos fármacos , Ciclopentanos/farmacologia , Resistência à Doença/efeitos dos fármacos , Oxilipinas/farmacologia , Actinidia/química , Actinidia/metabolismo , Ascomicetos/fisiologia , Quitinases/genética , Quitinases/metabolismo , Frutas/química , Frutas/metabolismo , Frutas/microbiologia , Expressão Gênica/efeitos dos fármacos , Peroxidação de Lipídeos/efeitos dos fármacos , Peroxidase/genética , Peroxidase/metabolismo , Fenóis/análise , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismo
2.
Anticancer Res ; 39(12): 6939-6943, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31810965

RESUMO

BACKGROUND/AIM: In spite of early detection, appoximately 15% of the small renal cell carcinomas (RCC) will develop metastasis within 5 years follow-up. The aim of this study was to identify new biomarkers to estimate the postoperative relapse of the most common conventional RCC. PATIENTS AND METHODS: Tissue multi arrays of conventional RCC without metastasis at the time of operation from a cohort of 634 patients were analysed by immunohistochemistry for expression of the chitinase 3-like protein 2 (CHI3L2). Cancer specific survival of patients was estimated with Kaplan-Meier analysis, univariate and multivariate Cox regression models. RESULTS: Kaplan-Meier analysis estimated a shorter cancer-free survival for patients with CHI3L2 positive tumors. In multivariate analysis, the CHI3L2 positivity associated with a 3.5 times higher risk for tumor relapse (p<0.001). CONCLUSION: Expression of CHI3L2 in tumor cells of conventional RCC define a group of patients at high risk for postoperative progression.


Assuntos
Carcinoma de Células Renais/metabolismo , Quitinases/metabolismo , Neoplasias Renais/metabolismo , Macrófagos/metabolismo , Recidiva Local de Neoplasia/metabolismo , Idoso , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/cirurgia , Quitinases/genética , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Renais/genética , Neoplasias Renais/cirurgia , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/genética , Recidiva Local de Neoplasia/cirurgia , Nefrectomia , Prognóstico , Análise de Sobrevida , Análise Serial de Tecidos , Regulação para Cima
3.
Microbiology ; 165(11): 1233-1244, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31526448

RESUMO

Serratia marcescens is a γ-Proteobacterium and an opportunistic animal and insect pathogen. The bacterium exhibits a complex extracellular protein 'secretome' comprising numerous enzymes, toxins and effector molecules. One component of the secretome is the 'chitinolytic machinery', which is a set of at least four chitinases that allow the use of insoluble extracellular chitin as sole carbon source. Secretion of the chitinases across the outer membrane is governed by the chiWXYZ operon encoding a holin/endopeptidase pair. Expression of the chiWXYZ operon is co-ordinated with the chitinase genes and is also bimodal, as normally only 1% of the population expresses the chitinolytic machinery. In this study, the role of the ChiR protein in chitinase production has been explored. Using live cell imaging and flow cytometry, ChiR was shown to govern the co-ordinated regulation of chiWXYZ with both chiA and chiC. Moreover, overexpression of chiR alone was able to increase the proportion of the cell population expressing chitinase genes to >60 %. In addition, quantitative label-free proteomic analysis of cells overexpressing chiR established that ChiR regulates the entire chitinolytic machinery. The proteomic experiments also revealed a surprising link between the regulation of the chitinolytic machinery and the production of proteins involved in the metabolism of nitrogen compounds such as nitrate and nitrite. The research demonstrates for the first time that ChiR plays a critical role in controlling bimodal gene expression in S. marcescens, and provides new evidence of a clear link between chitin breakdown and nitrogen metabolism.


Assuntos
Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Serratia marcescens/fisiologia , Proteínas de Bactérias/genética , Quitinases/genética , Citometria de Fluxo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Microscopia de Fluorescência , Mutação , Compostos de Nitrogênio/metabolismo , Óperon , Proteômica , Serratia marcescens/genética , Serratia marcescens/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
4.
Cell Mol Biol (Noisy-le-grand) ; 65(6): 73-80, 2019 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-31472050

RESUMO

Chitinases and N-acetyl-ß-glucosaminidase (NAG) are important in molting and growth of crustaceans. In ostracods, the genes encoding these enzymes have not been characterized. The aim of the present study was to clone the genes encoding chitinase (DsChi) and NAG (DsNAG) from the ostracod, Dolerocypris sinensis, elucidate the phylogenetic relationships between the cloned genes and known chitinolytic enzymes, and determine the expression patterns of these genes at different stages of growth in the presence of an environmental pollutant. The genes were amplified from the genomic DNA of the organism using polymerase chain reaction (PCR). The products from PCR were cloned and characterized with bioinformatics tools, and their expression patterns at different growth stages were determined using real-time quantitative PCR (qRT-PCR). Nine and five introns were identified in DsChi and DsNAG genes, respectively. When compared with protein sequences available in GenBank, chitinase from D. sinensis was most closely related to that of Macrobrachium nipponense (61 % homology). The NAG of D. sinensis was most closely related to that of Limulus polyphemus (55.6 % homology). Based on phylogenetic analysis of known chitinases from crustaceans and insects, the D. sinensis chitinase tightly clustered in the same branch with chitinases from species within the Malacostraca class. In contrast, NAG of D. sinensis was clustered with NAG from F. candida.The level of expression of DsChi mRNA was significantly higher than that of DsNAG throughout the period of growth (p < 0.05). Treatment of D. sinensis cells with fenoxycarb significantly downregulated the expressions of DsChi and DsNAG throughout the period of growth (p < 0.05). These results show that the protein products of DsChi and DsNAG possess remarkable biochemical properties characteristic of a chitinase and NAG, respectively.


Assuntos
Quitinases/genética , Crustáceos/enzimologia , Crustáceos/genética , Hexosaminidases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Quitinases/química , Clonagem Molecular , Crustáceos/crescimento & desenvolvimento , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genoma , Funções Verossimilhança , Fenilcarbamatos/farmacologia , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de DNA
5.
Pestic Biochem Physiol ; 159: 34-40, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31400782

RESUMO

The present study investigated the insecticidal activity of the different organic extracts from the entomopathogenic fungi, Cladosporium cladosporioides, Metarhizium anisopliae, Purpureocillium lilacinum, and Trichoderma longibrachiatum towards cotton aphid, Aphis gossypii. The methanol extracts from the mycelia and spores of C. cladosporioides and P. lilacinum exhibited the highest insecticidal activity against A. gossypii compared with other extracts, which LC50 values were recorded to be 57.60 and 94.18 ppm, respectively. The major constituents identified in both methanol extracts by GC-MS analysis were linoleic acid and palmitic acid. The methanol extracts of C. cladosporioides and P. lilacinum caused a voluminous increase in the total carbohydrates content of A. gossypii adults, while the total protein content was significantly decreased by both extracts. The activity of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were significantly reduced by methanol extracts. The P. lilacinum extract caused a considerable reduction in the activity of glutathione-S-transferase (GST), α- and ß-esterase by 28.9, 27.9 and 23.4%, respectively. Both extracts induced a significant increase in phenoloxidase and chitinase activity of A. gossypii adults. These results suggest that C. cladosporioides and P. lilacinum methanol extracts could be used as a promising approach for the management of A. gossypii in many economically crops.


Assuntos
Afídeos/efeitos dos fármacos , Gossypium/parasitologia , Inseticidas/farmacologia , Alanina Transaminase/genética , Alanina Transaminase/metabolismo , Animais , Quitinases/genética , Quitinases/metabolismo , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Resistência a Inseticidas/genética , Dose Letal Mediana , Metanol/química , Monofenol Mono-Oxigenase/genética , Monofenol Mono-Oxigenase/metabolismo
6.
World J Microbiol Biotechnol ; 35(7): 106, 2019 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-31267229

RESUMO

Xenorhabdus nematophila HB310 secreted the insecticidal protein toxin complex. Two chitinase genes, chi60 and chi70, were found in X. nematophila toxin complex locus. In order to clarify the function of two chitinases, chi60 and chi70 genes were cloned and expressed in Escherichia coli Transetta (DE3). As a result, we found that the Chi60 and Chi70 belonged to glycoside hydrolases (GH) family 18 with a molecular mass of 65 kDa and 78 kDa, respectively. When colloidal chitin was treated as the substrate, Chi60 and Chi70 were proved to have the highest enzymatic activity at pH 6.0 and 50 °C. Chi60 and Chi70 had obvious growth inhibition effect against the second larvae of Helicoverpa armigera with growth inhibiting rate of 81.99% and 90.51%. Chi70 had synergistic effect with the insecticidal toxicity of Bt Cry 1Ac, but the Chi60 had no synergistic effect with Bt Cry 1Ac. Chi60 and Chi70 showed antifungal activity against Alternaria brassicicola, Verticillium dahliae and Coniothyrium diplodiella. The results increased our understanding of the chitinases produced by X. nematophila and laid a foundation for further studies on the mechanism of the chitinases.


Assuntos
Antifúngicos/farmacologia , Quitinases/antagonistas & inibidores , Quitinases/genética , Quitinases/metabolismo , Xenorhabdus/metabolismo , Alternaria/efeitos dos fármacos , Animais , Ascomicetos/efeitos dos fármacos , Quitina/metabolismo , Quitinases/classificação , Clonagem Molecular , Sinergismo Farmacológico , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Expressão Gênica , Glicosídeo Hidrolases/genética , Concentração de Íons de Hidrogênio , Inseticidas/metabolismo , Inseticidas/farmacologia , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Peso Molecular , Mariposas/efeitos dos fármacos , Mariposas/crescimento & desenvolvimento , Micotoxinas/genética , Micotoxinas/metabolismo , Filogenia , Domínios Proteicos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Temperatura Ambiente , Verticillium/efeitos dos fármacos , Xenorhabdus/genética
7.
Int J Mol Sci ; 20(14)2019 Jul 16.
Artigo em Inglês | MEDLINE | ID: mdl-31315176

RESUMO

In this study we cloned a chitinase gene (SmchiC), from Serratia marcescens isolated from the corpse of a Diatraea magnifactella lepidopteran, which is an important sugarcane pest. The chitinase gene SmchiC amplified from the S. marcescens genome was cloned into the transformation vector p2X35SChiC and used to transform tobacco (Nicotiana tabacum L. cv Petit Havana SR1). The resistance of these transgenic plants to the necrotrophic fungus Botrytis cinerea and to the pest Spodoptera frugiperda was evaluated: both the activity of chitinase as well as the resistance against B. cinerea and S. frugiperda was significantly higher in transgenic plants compared to the wild-type.


Assuntos
Proteínas de Bactérias/genética , Quitinases/genética , Resistência à Doença/genética , Serratia marcescens/genética , Tabaco/genética , Transgenes , Animais , Proteínas de Bactérias/metabolismo , Botrytis/patogenicidade , Quitinases/metabolismo , Spodoptera/patogenicidade , Tabaco/microbiologia , Tabaco/parasitologia
8.
Carbohydr Res ; 483: 107747, 2019 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-31349143

RESUMO

A new extracellular chitinase (called ChiA-Pt70) was produced and purified from a newly isolated Paenibacillus timonensis strain LK-DZ15. The maximum chitinase activity recorded after 44-h of incubation at 30 °C was 11,500 U/mL. Pure enzyme was obtained after ammonium sulphate precipitation (40-70%) followed by sequential column chromatographies on fast performance liquid chromatography (FPLC) and high performance liquid chromatography (HPLC). Based on matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF/MS) analysis, the purified enzyme is a monomer with a molecular mass of 70,166.11 kDa. The sequence of the 25 NH2-terminal residues of the mature ChiA-70 showed high homology with Paenibacillus GH-18 chitinases family. Optimal activity was achieved at pH 4.5 and 80 °C. The pure enzyme was completely inhibited by p-chloromercuribenzoic acid (p-CMB), 5,5'-dithio-bis-2-nitro benzoic acid (DTNB), and N-ethylmaleimide (NEM). Chitinase activity was high on colloidal chitin, chitin azure, glycol chitin, glycol chitosane, chitotriose, and chito-oligosaccharide while it did not hydrolyse chitibiose and amylose. Furthermore, thin-layer chromatography (TLC) analysis from enzymatic catalyzed hydrolysis of colloidal chitin showed that ChiA-Pt70 acted as an endo-splitting enzyme. Its Km and kcat values were 0.611 mg colloidal chitin/mL and 87,800 s-1, respectively. Interestingly, its catalytic efficiency was higher than those of chitinases ChiA-Mt45 from Melghiribacillus thermohalophilus strain Nari2AT, ChiA-Hh59 from Hydrogenophilus hirchii strain KB-DZ44, Chitodextrinase® from Streptomyces griseus, and N-acetyl-ß-glucosaminidase® from Trichoderma viride. Therefore, ChiA-Pt70 exhibited remarkable biochemical properties suggesting that it is suitable for the enzymatic degradation of chitin.


Assuntos
Quitinases/genética , Quitinases/isolamento & purificação , Paenibacillus/enzimologia , Argélia , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Quitinases/metabolismo , Cromatografia Líquida de Alta Pressão , Concentração de Íons de Hidrogênio , Peso Molecular , Paenibacillus/classificação , Paenibacillus/genética , Filogenia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , Termodinâmica
9.
Genes (Basel) ; 10(6)2019 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-31234426

RESUMO

Chitinase is a kind of hydrolase with chitin as a substrate and is proposed to play an essential role in plant defense system by functioning against fungal pathogens through degrading chitin. Recent studies indicated chitinase is also involved in abiotic stress response in plants, helping plants to survive in stressful environments. A. nanus, a rare evergreen broad-leaved shrub distrusted in deserts in Central Asia, exhibits a high level of tolerance to drought and low temperature stresses. To identify the chitinase gene involved in drought and low temperature responses in A. nanus, we performed genome-wide identification, classification, sequence alignment, and spatio-temporal gene expression analysis of the chitinases in A. nanus under osmotic and low temperature stress. A total of 32 chitinase genes belonging to glycosyl hydrolase 18 (GH18) and GH19 families were identified from A. nanus. Class III chitinases appear to be amplified quantitatively in A. nanus, and their genes carry less introns, indicating their involvement in stress response in A. nanus. The expression level of the majority of chitinases varied in leaves, stems, and roots, and regulated under environmental stress. Some chitinases, such as EVM0022783, EVM0020238, and EVM0003645, are strongly induced by low temperature and osmotic stress, and the MYC/ICE1 (inducer of CBF expression 1) binding sites in promoter regions may mediate the induction of these chitinases under stress. These chitinases might play key roles in the tolerance to these abiotic stress in A. nanus and have potential for biotechnological applications. This study provided important data for understanding the biological functions of chitinases in A. nanus.


Assuntos
Quitinases/genética , Resposta ao Choque Frio/genética , Fabaceae/genética , Filogenia , Quitinases/classificação , Secas , Fabaceae/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Genoma de Planta/genética , Pressão Osmótica/fisiologia , Folhas de Planta/genética , Alinhamento de Sequência , Estresse Fisiológico/genética
10.
Bull Entomol Res ; 109(6): 741-751, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31113496

RESUMO

Chitinase is responsible for insect chitin hydrolyzation, which is a key process in insect molting and pupation. However, little is known about the chitinase of Spodoptera exigua (SeChi). In this study, based on the SeChi gene (ADI24346) identified in our laboratory, we constructed the recombinant baculovirus P-Chi for the expression of recombinant SeChi (rSeChi) in Hi5 cells. The rSeChi was purified by chelate affinity chromatography, and the purified protein showed activity comparable with that of a commercial SgChi, suggesting that we harvested active SeChi for the first time. The purified protein was subsequently tested for enzymatic properties and revealed to exhibit its highest activity at pH 8 and 40 C. Using homology modeling and molecular docking techniques, the three-dimensional model of SeChi was constructed and screened for inhibitors. In two rounds of screening, twenty compounds were selected. With the purified rSeChi, we tested each of the twenty compounds for inhibitor activity against rSeChi, and seven compounds showed obvious activity. This study provided new information for the chitinase of beet armyworm and for chitinase inhibitor development.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Spodoptera/enzimologia , Animais , Linhagem Celular , Quitinases/genética , Quitinases/isolamento & purificação , Quitinases/metabolismo , Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Simulação de Acoplamento Molecular , Mariposas , Spodoptera/genética
11.
Int J Biol Macromol ; 134: 882-890, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31108147

RESUMO

Chitotriosidase (Chit1) and acidic mammalian chitinase (AMCase) have been implicated in food processing and various pathophysiological conditions such as chronic inflammatory diseases. By combination of the colorimetric analysis and fluorophore-assisted carbohydrate electrophoresis (FACE) method, we directly compared the chitinolytic properties of mouse Chit1 and AMCase and determined their combinatory effects in artificial and natural chitin substrates processing. Chit1 and AMCase display different dynamics of chitinolytic properties through acidic to neutral conditions. At pH2.0, the activity of AMCase was higher than that of Chit1 and stronger or comparable with that of Serratia marcescens chitinase B, a well-characterized bacterium chitinase. Changes of degradation products using different substrates indicate that AMCase and Chit1 have diverse properties under various pH conditions. Exposure of the chitin substrates to both Chit1 and AMCase did not indicate any mutual interference of these enzymes and showed no synergistic effect, in contrast to observations regarding some bacterial chitinases. Our results suggest that Chit1 and AMCase have no synergistic effect under physiological conditions.


Assuntos
Quitina/química , Quitinases/química , Hexosaminidases/química , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Quitinases/genética , Colorimetria , Concentração de Íons de Hidrogênio , Hidrólise , Camundongos , Peso Molecular , Proteínas Recombinantes , Especificidade por Substrato
12.
Mol Plant Microbe Interact ; 32(9): 1121-1133, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31039081

RESUMO

ChiIV3, a chitinase of pepper (Capsicum annuum), stimulates cell death in pepper plants. However, there are only scarce reports on its role in resistance against bacterial wilt disease such as that caused by Ralstonia solanacearum and their transcriptional regulation. In this study, the silencing of ChiIV3 in pepper plants significantly reduced the resistance to R. solanacearum. The transcript of ChiIV3 was induced by R. solanacearum inoculation (RSI) as well as exogenous application of methyl jasmonate and abscisic acid. The bioinformatics analysis revealed that the ChiIV3 promoter consists of multiple stress-related cis elements, including six W-boxes and one MYB1AT. With the 5' deletion assay in the ChiIV3 promoter, the W4-box located from -640 to -635 bp was identified as the cis element that is required for the response to RSI. In addition, the W4-box element was shown to be essential for the binding of the ChiIV3 promoter by the WRKY40 transcription factor, which is known to positively regulate the defense response to R. solanacearum. Site-directed mutagenesis in the W4-box sequence impaired the binding of WRKY40 to the ChiIV3 promoter. Subsequently, the transcription of ChiIV3 decreased in WRKY40-silenced pepper plants. These results demonstrated that the expression of the defense gene ChiIV3 is controlled through multiple modes of regulation, and WRKY40 directly binds to the W4-box element of the ChiIV3 promoter region for its transcriptional regulation.


Assuntos
Capsicum , Quitinases , Resistência à Doença , Ralstonia solanacearum , Fatores de Transcrição , Capsicum/enzimologia , Capsicum/genética , Capsicum/microbiologia , Quitinases/genética , Quitinases/metabolismo , Resistência à Doença/genética , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Humanos , Mutagênese Sítio-Dirigida , Doenças das Plantas/microbiologia , Proteínas de Plantas , Ligação Proteica/genética , Ralstonia solanacearum/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Int J Biol Macromol ; 134: 113-121, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31034902

RESUMO

In this study, a chitinase gene, Chit46 from a mycoparasitic fungus Trichoderma harzianum was successfully expressed in Pichia pastoris with a high heterologous chitinase production of 31.4 U/mL, much higher than the previous reports. The active center and substrate binding pocket of the recombinant Chit46 (rChit46) were analyzed and the effects of pH, temperature, metal ions and glycosylation on its activity were tested. rChit46 effectively hydrolyzed colloidal chitin with a high conversion rate of 80.5% in 3 h and the chitin hydrolysates were mainly composed of (GlcNAc)2 (94.8%), which make it a good candidate for the green recycling of chitinous waste. rChit46 could also significantly inhibit growth of the phytopathogenic fungus Botrytis cinerea, which endowed it with the potential as a biocontrol agent.


Assuntos
Quitina/química , Quitinases/genética , Quitinases/metabolismo , Coloides , Trichoderma/enzimologia , Trichoderma/genética , Adsorção , Antifúngicos/química , Antifúngicos/farmacologia , Quitina/metabolismo , Quitinases/química , Cromatografia Líquida de Alta Pressão , Ativação Enzimática , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Proteínas Recombinantes , Especificidade por Substrato , Temperatura Ambiente
14.
Dokl Biochem Biophys ; 484(1): 29-32, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31012007

RESUMO

Fifteen chitinases of classes I-V were identified in the transcriptomes of pitchers and adult leaves of the carnivorous plant Nepenthes sp. Ten of these chitinases were identified for the first time, including the chitinases of classes II and V. The expression levels of all found chitinase genes in leaves and at three stages of pitcher development were determined. The maximum level of transcriptional activity in an open pitcher was observed for the genes encoding chitinase NChi4 (class II) and its isoforms. The expression levels of these genes significantly increased as the pitcher developed. In addition, for the first time, transcription of the genes encoding chitinases of all five classes was detected in the leaves of this plant.


Assuntos
Caryophyllales , Quitinases , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Genes de Plantas , Proteínas de Plantas , Caryophyllales/enzimologia , Caryophyllales/genética , Quitinases/biossíntese , Quitinases/genética , Isoenzimas/biossíntese , Isoenzimas/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética
15.
Enzyme Microb Technol ; 126: 50-61, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31000164

RESUMO

The biocontrol activity of some soil strains of Chromobacterium sp. against pathogenic fungi has been attributed to secreted chitinases. The aim of this work was to characterize biochemically a recombinant chitinase (CvChi47) from C. violaceum ATCC 12472 and to investigate its effects on phytopathogenic fungi. CvChi47 is a modular enzyme with 450 amino acid residues, containing a type I signal peptide at the N-terminal region, followed by one catalytic domain belonging to family 18 of the glycoside hydrolases, and two type-3 chitin-binding domains at the C-terminal end. The recombinant enzyme was expressed in Escherichia coli as a His-tagged protein and purified to homogeneity. The native signal peptide of CvChi47 was used to direct its secretion into the culture medium, from where the recombinant product was purified by affinity chromatography on chitin and immobilized metal. The purified protein showed an apparent molecular mass of 46 kDa, as estimated by denaturing polyacrylamide gel electrophoresis, indicating the removal of the signal peptide. CvChi47 was a thermostable protein, retaining approximately 53.7% of its activity when heated at 100 °C for 1 h. The optimum hydrolytic activity was observed at 60 °C and pH 5. The recombinant chitinase inhibited the conidia germination of the phytopathogenic fungi Fusarium oxysporum and F. guttiforme, hence preventing mycelial growth. Furthermore, atomic force microscopy experiments revealed a pronounced morphological alteration of the cell surface of conidia incubated with CvChi47 in comparison to untreated cells. Taken together, these results show the potential of CvChi47 as a molecular tool to control plant diseases caused by these Fusarium species.


Assuntos
Antifúngicos/farmacologia , Quitinases/metabolismo , Chromobacterium/enzimologia , Fusarium/crescimento & desenvolvimento , Doenças das Plantas/prevenção & controle , Proteínas Recombinantes/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Quitinases/química , Quitinases/genética , Clonagem Molecular , Estabilidade Enzimática , Fusarium/efeitos dos fármacos , Doenças das Plantas/microbiologia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Homologia de Sequência , Esporos Fúngicos/efeitos dos fármacos , Esporos Fúngicos/crescimento & desenvolvimento , Temperatura Ambiente
16.
Carbohydr Res ; 478: 1-9, 2019 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-31005672

RESUMO

Chitooligosaccharides (COS), the depolymerization products of chitin, have many potential applications in agriculture and medicine since they induce immunostimulating effects and disease protective responses. Most of their biological activities require degrees of polymerization (DP) larger than the tetrasaccharide, but structurally well-defined COS with DP larger than six are difficult to produce due to their high insolubility and complex isolation from chitin hydrolysates. Enzymatic synthesis by exploiting the transglycosylation activity of chitinases offers a potential strategy for the assembly of oligomers in the range of bioactive DPs. We here explore the glycosynthase-like activity of six GH18 chitinases from bacterial and archaeal origin by mutating the catalytic assisting residue in the substrate-assisted mechanism of this enzyme family. The alanine mutants at the assisting residue have a significant, but not essential, effect on the hydrolase activity. We studied the ability of the alanine mutants at the assisting residue to catalyze the polymerization of an oxazoline derivative as donor substrate, selecting the oxazoline of pentaacetylchitopentaose (DP5ox) with the aim of obtaining larger oligomers/polymers that, being insoluble, might be resistant to further reactions by the hydrolytically compromised mutant enzymes. For all the enzymes, insoluble polymeric material was obtained, with DP10 as major component, but other COS with different DPs were also obtained, limiting the practical application to produce oligomers/polymers with a defined DP. The balance between the residual hydrolase activity of the mutant enzymes and the solubility/precipitation kinetics still lead to hydrolysis and/or transglycosylation reactions on the newly formed products. From the selected enzymes, the Thermococcus kodakaraensis ChiA D1022A mutant gave the best results, with the formation of insoluble polymers in 45% yield (w/w) and containing about 55% of the target DP10 product.


Assuntos
Quitina/análogos & derivados , Quitinases/genética , Quitinases/metabolismo , Biocatálise , Configuração de Carboidratos , Quitina/biossíntese , Quitina/química , Mutação , Polimerização
17.
Int J Biol Macromol ; 132: 1235-1243, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-30980875

RESUMO

As the main component of the fungal cell wall, chitin has been regarded as an optimal molecular target for the biocontrol of plant-pathogenic fungi. In this study, the chitin hydrolase CcCti1, which belongs to the glycoside hydrolase family 18 (GH 18) and exhibits potential antifungal activity, was identified from Corallococcus sp. EGB. CcCti1 lacks a fibronectin type-III (FN3) domain that is present in similar enzymes from most genera of myxobacteria, indicating that CcCti1 may have acquired chitinase activity due to the FN3 domain deletion during myxobacterial evolution. CcCti1 was expressed in Escherichia coli BL21 (DE3) with a specific activity of up to 10.5 U/µmol with colloidal chitin as the substrate. Product analysis showed that CcCti1 could hydrolyze chitin into N-acetylated chitohexaose (GlcNAc)6 as the major product, in addition to chitooligosaccharides. The analysis of biochemical properties indicated that the CBD and FN3 domains in CcCti1 determine the substrate affinity and pH stability. Otherwise, CcCti1 exhibited efficient biocontrol activity against the plant pathogen Magnaporthe oryzae in a dose-dependent manner, inhibiting the conidia germination and appressoria formation at a concentration of 0.08 mg/mL. Overall, the chitohexaose-producing chitinase CcCti1 with hydrolytic features may find potential application in chitin conversion and biocontrol of fungal plant diseases.


Assuntos
Antifúngicos/metabolismo , Antifúngicos/farmacologia , Quitinases/genética , Quitinases/farmacologia , Myxococcales/efeitos dos fármacos , Sequência de Aminoácidos , Antifúngicos/química , Quitinases/química , Clonagem Molecular , Evolução Molecular , Hidrólise , Filogenia , Domínios Proteicos
18.
Emerg Microbes Infect ; 8(1): 17-28, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30866756

RESUMO

An outbreak with a remarkable Listeria monocytogenes clone causing 163 cases of non-invasive listeriosis occurred in Germany in 2015. Core genome multi locus sequence typing grouped non-invasive outbreak isolates and isolates obtained from related food samples into a single cluster, but clearly separated genetically close isolates obtained from invasive listeriosis cases. A comparative genomic approach identified a premature stop codon in the chiB gene, encoding one of the two L. monocytogenes chitinases, which clustered with disease outcome. Correction of this premature stop codon in one representative gastroenteritis outbreak isolate restored chitinase production, but effects in infection experiments were not found. While the exact role of chitinases in virulence of L. monocytogenes is still not fully understood, our results now clearly show that ChiB-derived activity is not required to establish L. monocytogenes gastroenteritis in humans. This limits a possible role of ChiB in human listeriosis to later steps of the infection.


Assuntos
Quitinases/genética , Surtos de Doenças , Gastroenterite/microbiologia , Listeria monocytogenes/classificação , Listeria monocytogenes/isolamento & purificação , Listeriose/epidemiologia , Adolescente , Adulto , Proteínas de Bactérias/genética , Técnicas de Tipagem Bacteriana , Células CACO-2 , Criança , Pré-Escolar , Códon de Terminação , Feminino , Microbiologia de Alimentos , Gastroenterite/epidemiologia , Genômica , Alemanha/epidemiologia , Células HeLa , Células Hep G2 , Humanos , Lactente , Listeria monocytogenes/enzimologia , Listeria monocytogenes/patogenicidade , Masculino , Pessoa de Meia-Idade , Tipagem de Sequências Multilocus , Filogenia , Fatores de Virulência/genética , Adulto Jovem
19.
J Agric Food Chem ; 67(13): 3575-3582, 2019 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-30865442

RESUMO

Insect chitinases play an indispensable role in shedding old cuticle during molting. Targeting chitinase inhibition is a promising pest control strategy. Of ChtI, a chitinase from the destructive insect pest Ostrinia furnacalis (Asian corn borer), has been suggested as a potential target for designing green pesticides. A 4,5,6,7-tetrahydrobenzo[ b]thiophene-3-carboxylate scaffold was previously obtained, and further derivatization generated the lead compound 1 as Of ChtI inhibitor. Here, based on the predicted binding mode of compound 1, the pocket-based lead optimization strategy was applied. A series of analogues was synthesized, and their inhibitory activities against Of ChtI were evaluated. Compound 8 with 6- tert-pentyl showed preferential inhibitory activity with a Ki value of 0.71 µM. Their structure-activity relationships suggested that the compound with larger steric hindrance at the 6-nonpolar group was essential for inhibitory activity due to its stronger interactions with surrounding amino acids. This work provides a strategy for designing potential chitinase inhibitors.


Assuntos
Quitinases/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas de Insetos/antagonistas & inibidores , Inseticidas/química , Mariposas/enzimologia , Animais , Domínio Catalítico , Quitinases/genética , Quitinases/metabolismo , Desenho de Drogas , Inibidores Enzimáticos/farmacologia , Proteínas de Insetos/genética , Proteínas de Insetos/metabolismo , Inseticidas/farmacologia , Cinética , Mariposas/química , Mariposas/efeitos dos fármacos , Domínios Proteicos
20.
Curr Microbiol ; 76(5): 566-574, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30820638

RESUMO

Burkholderia cenocepacia TAtl-371 was isolated from the rhizosphere of a tomato plant growing in Atlatlahucan, Morelos, Mexico. This strain exhibited a broad antimicrobial spectrum against bacteria, yeast, and fungi. Here, we report and describe the improved, high-quality permanent draft genome of B. cenocepacia TAtl-371, which was sequenced using a combination of PacBio RS and PacBio RS II sequencing methods. The 7,496,106 bp genome of the TAtl-371 strain is arranged in three scaffolds, contains 6722 protein-coding genes, and 99 RNA only-encoding genes. Genome analysis revealed genes related to biosynthesis of antimicrobials such as non-ribosomal peptides, siderophores, chitinases, and bacteriocins. Moreover, analysis of bacterial growth on different carbon and nitrogen sources shows that the strain retains its antimicrobial ability.


Assuntos
Antibiose , Burkholderia cenocepacia/genética , Complexo Burkholderia cepacia , Carbono/metabolismo , Genoma Bacteriano , Nitrogênio/metabolismo , Bacteriocinas/genética , Burkholderia cenocepacia/isolamento & purificação , Quitinases/genética , Lycopersicon esculentum/microbiologia , México , Rizosfera , Análise de Sequência de DNA , Sideróforos/genética , Microbiologia do Solo
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