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1.
J Agric Food Chem ; 65(2): 435-444, 2017 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-27997168

RESUMO

This study aimed to evaluate the fermentation in the large intestine of indigestible dietary protein sources from animal, insect, and plant origin using an in vitro model of the pig's gastrointestinal tract. Protein sources were used raw and after a cooking treatment. Results showed that the category of the ingredient (meats, insects, or grain legumes) exerts a stronger impact on enzymatic digestibility, fermentation patterns, and bacterial metabolites such as short-chain fatty acids (SCFA) and hydrogen sulfide (H2S) than the cooking treatment. The digestibility and the fermentation characteristics of insects were more affected by the cooking procedure than the other categories. Per gram of consumed food, ingredients from animal origin, namely, meats and insects, were associated with fewer fermentation end-products (gas, H2S, SCFA) than ingredients from plant origin, which is related to their higher small intestinal digestibility.


Assuntos
Culinária , Fabaceae/metabolismo , Trato Gastrointestinal/metabolismo , Insetos/metabolismo , Ração Animal , Animais , Digestão , Fermentação , Sulfeto de Hidrogênio/análise , Sulfeto de Hidrogênio/metabolismo , Proteínas de Insetos/metabolismo , Proteínas de Insetos/farmacocinética , Absorção Intestinal , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Carne , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacocinética , Sus scrofa/metabolismo
2.
Environ Sci Pollut Res Int ; 22(6): 4369-82, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25300185

RESUMO

The purpose of this work was the isolation and cultivation of cellulolytic and xylanolytic microorganisms extracted from the gut of the lower termite Reticulitermes santonensis. Microcrystalline cellulose (with and without lignin) and beech wood xylan were used as diets instead of poplar wood in order to select cellulose and hemicellulose-degrading fungi. The strain Sarocladium kiliense (Acremonium kiliense) CTGxxyl was isolated from the termites fed on xylan, while the strain Trichoderma virens CTGxAviL was isolated from the termites fed on cellulose (with and without lignin). Both molds were cultivated in liquid media containing different substrates: agro-residues or purified polymers. S. kiliense produced maximal ß-glucosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase and endo-1,4-ß-D-xylanase activities of 0.103, 3.99, 0.53, and 40.8 IU/ml, respectively. T. virens produced maximal ß-xylosidase, endo-1,4-ß-D-glucanase, exo-1,4-ß-D-glucanase, and endo-1,4-ß-D-xylanase activities of 0.38, 1.48, 0.69, and 426 IU/ml. The cellulase and the xylanase of S. kiliense, less common than T. virens, were further investigated. The optimal activity of the xylanase was observed at pH 9-10 at 60 °C. The cellulase showed its maximal activity at pH 10, 70 °C. Zymography identified different xylanases produced by both molds, and some fragment sizes were highlighted: 35, 100, and 170 kDa for S. kiliense and 20, 40, 80, and 170 kDa for T. virens. In both cases, endo-1,4-ß-D-xylanase activities were confirmed through mass spectrometry.


Assuntos
Celulose/metabolismo , Trato Gastrointestinal/microbiologia , Hypocreales/isolamento & purificação , Isópteros/microbiologia , Trichoderma/isolamento & purificação , Xilanos/metabolismo , Animais , Técnicas de Cultura de Células , Celulase , Celulases/metabolismo , Concentração de Íons de Hidrogênio , Hypocreales/crescimento & desenvolvimento , Hypocreales/metabolismo , Temperatura , Trichoderma/crescimento & desenvolvimento , Trichoderma/metabolismo , Xilosidases/metabolismo
3.
Front Microbiol ; 6: 1487, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26734000

RESUMO

Bacteria degrading algal polysaccharides are key players in the global carbon cycle and in algal biomass recycling. Yet the water column, which has been studied largely by metagenomic approaches, is poor in such bacteria and their algal-polysaccharide-degrading enzymes. Even more surprisingly, the few published studies on seaweed-associated microbiomes have revealed low abundances of such bacteria and their specific enzymes. However, as macroalgal cell-wall polysaccharides do not accumulate in nature, these bacteria and their unique polysaccharidases must not be that uncommon. We, therefore, looked at the polysaccharide-degrading activity of the cultivable bacterial subpopulation associated with Ascophyllum nodosum. From A. nodosum triplicates, 324 bacteria were isolated and taxonomically identified. Out of these isolates, 78 (~25%) were found to act on at least one tested algal polysaccharide (agar, ι- or κ-carrageenan, or alginate). The isolates "active" on algal-polysaccharides belong to 11 genera: Cellulophaga, Maribacter, Algibacter, and Zobellia in the class Flavobacteriia (41) and Pseudoalteromonas, Vibrio, Cobetia, Shewanella, Colwellia, Marinomonas, and Paraglaceciola in the class Gammaproteobacteria (37). A major part represents likely novel species. Different proportions of bacterial phyla and classes were observed between the isolated cultivable subpopulation and the total microbial community previously identified on other brown algae. Here, Bacteroidetes and Gammaproteobacteria were found to be the most abundant and some phyla (as Planctomycetes and Cyanobacteria) frequently encountered on brown algae weren't identified. At a lower taxonomic level, twelve genera, well-known to be associated with algae (with the exception for Colwellia), were consistently found on all three A. nosodum samples. Even more interesting, 9 of the 11 above mentioned genera containing polysaccharolytic isolates were predominant in this common core. The cultivable fraction of the bacterial community associated with A. nodosum is, thus, significantly enriched in macroalgal-polysaccharide-degrading bacteria and these bacteria seem important for the seaweed holobiont even though they are under-represented in alga-associated microbiome studies.

4.
Microb Ecol ; 68(4): 822-33, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25008985

RESUMO

In soils, bacteria are very abundant and diverse. They are involved in various agro-ecosystem processes such as the nitrogen cycle, organic matter degradation, and soil formation. Yet, little is known about the distribution and composition of bacterial communities through the soil profile, particularly in agricultural soils, as most studies have focused only on topsoils or forest and grassland soils. In the present work, we have used bar-coded pyrosequencing analysis of the V3 region of the 16S rRNA gene to analyze bacterial diversity in a profile (depths 10, 25, and 45 cm) of a well-characterized field of winter wheat. Taxonomic assignment was carried out with the Ribosomal Database Project (RDP) Classifier program with three bootstrap scores: a main run at 0.80, a confirmation run at 0.99, and a run at 0 to gain information on the unknown bacteria. Our results show that biomass and bacterial quantity and diversity decreased greatly with depth. Depth also had an impact, in terms of relative sequence abundance, on 81 % of the most represented taxonomic ranks, notably the ranks Proteobacteria, Bacteroidetes, Actinobacteridae, and Acidobacteria. Bacterial community composition differed more strongly between the topsoil (10 and 25 cm) and subsoil (45 cm) than between levels in the topsoil, mainly because of shifts in the carbon, nitrogen, and potassium contents. The subsoil also contained more unknown bacteria, 53.96 % on the average, than did the topsoil, with 42.06 % at 10 cm and 45.59 % at 25 cm. Most of these unknown bacteria seem to belong to Deltaproteobacteria, Actinobacteria, Rhizobiales, and Acidobacteria.


Assuntos
Bactérias/genética , Biodiversidade , Microbiota , Estações do Ano , Microbiologia do Solo , Solo/química , Triticum/microbiologia , Bactérias/isolamento & purificação , Bélgica , Código de Barras de DNA Taxonômico , DNA Bacteriano/genética , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Reação em Cadeia da Polimerase em Tempo Real , Triticum/crescimento & desenvolvimento
5.
Appl Environ Microbiol ; 80(16): 4958-67, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24907332

RESUMO

A metagenomic library was constructed from microorganisms associated with the brown alga Ascophyllum nodosum. Functional screening of this library revealed 13 novel putative esterase loci and two glycoside hydrolase loci. Sequence and gene cluster analysis showed the wide diversity of the identified enzymes and gave an idea of the microbial populations present during the sample collection period. Lastly, an endo-ß-1,4-glucanase having less than 50% identity to sequences of known cellulases was purified and partially characterized, showing activity at low temperature and after prolonged incubation in concentrated salt solutions.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Glicosídeo Hidrolases/química , Glicosídeo Hidrolases/genética , Microbiota , Phaeophyceae/microbiologia , Alga Marinha/microbiologia , Bactérias/classificação , Bactérias/genética , Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Temperatura Baixa , Estabilidade Enzimática , Glicosídeo Hidrolases/metabolismo , Metagenômica , Dados de Sequência Molecular , Filogenia , Cloreto de Sódio/metabolismo
6.
Molecules ; 19(4): 4578-94, 2014 Apr 11.
Artigo em Inglês | MEDLINE | ID: mdl-24731986

RESUMO

Hindgut homogenates of the termite Reticulitermes santonensis were incubated with carboxymethyl cellulose (CMC), crystalline celluloses or xylan substrates. Hydrolysates were analyzed with matrix-assisted laser desorption/ionization coupled to time-of-flight mass spectrometry (MALDI-TOF MS). The method was first set up using acid hydrolysis analysis to characterize non-enzymatic profiles. Commercial enzymes of Trichoderma reesei or T. longibrachiatum were also tested to validate the enzymatic hydrolysis analysis. For CMC hydrolysis, data processing and visual display were optimized to obtain comprehensive profiles and allow rapid comparison and evaluation of enzymatic selectivity, according to the number of substituents of each hydrolysis product. Oligosaccharides with degrees of polymerization (DPs) ranging from three to 12 were measured from CMC and the enzymatic selectivity was demonstrated. Neutral and acidic xylo-oligosaccharides with DPs ranging from three to 11 were measured from xylan substrate. These results are of interest for lignocellulose biomass valorization and demonstrated the potential of termites and their symbiotic microbiota as a source of interesting enzymes for oligosaccharides production.


Assuntos
Celulose/análogos & derivados , Dextrinas/química , Intestinos/química , Isópteros/química , Oligossacarídeos/química , Animais , Carboximetilcelulose Sódica/química , Celulose/química , Misturas Complexas/química , Proteínas Fúngicas/química , Hidrólise , Proteínas de Insetos/química , Intestinos/enzimologia , Isópteros/enzimologia , Trichoderma/química , Trichoderma/enzimologia , Xilanos/química
7.
Appl Microbiol Biotechnol ; 98(7): 2917-35, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24562178

RESUMO

Marine microorganisms play key roles in every marine ecological process, hence the growing interest in studying their populations and functions. Microbial communities on algae remain underexplored, however, despite their huge biodiversity and the fact that they differ markedly from those living freely in seawater. The study of this microbiota and of its relationships with algal hosts should provide crucial information for ecological investigations on algae and aquatic ecosystems. Furthermore, because these microorganisms interact with algae in multiple, complex ways, they constitute an interesting source of novel bioactive compounds with biotechnological potential, such as dehalogenases, antimicrobials, and alga-specific polysaccharidases (e.g., agarases, carrageenases, and alginate lyases). Here, to demonstrate the huge potential of alga-associated organisms and their metabolites in developing future biotechnological applications, we first describe the immense diversity and density of these microbial biofilms. We further describe their complex interactions with algae, leading to the production of specific bioactive compounds and hydrolytic enzymes of biotechnological interest. We end with a glance at their potential use in medical and industrial applications.


Assuntos
Bactérias/classificação , Bactérias/isolamento & purificação , Biodiversidade , Produtos Biológicos/metabolismo , Biotecnologia/métodos , Alga Marinha/microbiologia , Bactérias/metabolismo , Alga Marinha/fisiologia , Simbiose
8.
J Ind Microbiol Biotechnol ; 41(3): 479-88, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24414432

RESUMO

New ß-glucosidase activities were identified by screening metagenomic libraries constructed with DNA isolated from the topsoil of a winter wheat field. Two of the corresponding proteins, displaying an unusual preference for alkaline conditions, were selected for purification by Ni-NTA chromatography. AS-Esc6, a 762-amino-acid enzyme belonging to glycoside hydrolase family 3, proved to be a mesophilic aryl-ß-glucosidase with maximal activity around pH 8 and 40 °C. A similar pH optimum was found for AS-Esc10, a 475-amino-acid GH1-family enzyme, but this enzyme remained significantly active across a wider pH range and was also markedly more stable than AS-Esc6 at pH greater than 10. AS-Esc10 was found to degrade cellobiose and diverse aryl glycosides, with an optimal temperature of 60 °C and good stability up to 50 °C. Unlike AS-Esc6, which showed a classically low inhibitory constant for glucose (14 mM), AS-Esc10 showed enhanced activity in the presence of molar concentrations of glucose. AS-Esc10 was highly tolerant to hydrogen peroxide and also to sodium dodecyl sulfate, this being indicative of kinetic stability. This unique combination of properties makes AS-Esc10 a particularly promising candidate whose potential in biotechnological applications is worth exploring further.


Assuntos
Metagenômica , Solo/química , beta-Glucosidase/genética , beta-Glucosidase/isolamento & purificação , Detergentes/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Temperatura , beta-Glucosidase/química , beta-Glucosidase/metabolismo
9.
World J Microbiol Biotechnol ; 30(5): 1655-60, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24353041

RESUMO

The aim of this work was to isolate enzyme-producing microorganisms from the tract of the termite Reticulitermes santonensis. The microorganisms were extracted from the guts and anaerobic (CO2 or CO2/H2) and micro-aerobic atmospheres were used to stimulate growth. Three different strategies were tried out. First, the sample was spread on Petri dishes containing solid media with carboxymethylcellulose, microcrystalline cellulose or cellobiose. This technique allowed us to isolate two bacteria: Streptomyces sp. strain ABGxAviA1 and Pseudomonas sp. strain ABGxCellA. The second strategy consisted in inoculating a specific liquid medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. The samples were then spread on Petri dishes with the same specific medium containing carboxymethylcellulose, microcrystalline cellulose, or cellobiose. This led to the isolation of the mold Aspergillus sp. strain ABGxAviA2. Finally, the third strategy consisted in heating the first culture and spreading samples on agar plates containing rich medium. This led to the isolation of the bacterium Bacillus subtilis strain ABGx. All those steps were achieved in controlled atmospheres. The four enzyme-producing strains which were isolated were obtained by using a micro-aerobic atmosphere. Later, enzymatic assays were performed on the four strains. Streptomyces sp. strain ABGxAviA1 was found to produce only amylase, while Pseudomonas sp. strain ABGxCellA was found to produce ß-glucosidase as well. Aspergillus sp. strain ABGxAviA2 showed ß-glucosidase, amylase, cellulase, and xylanase activities. Finally, B. subtilis strain ABGx produced xylanase and amylase.


Assuntos
Aspergillus/enzimologia , Aspergillus/isolamento & purificação , Bactérias/enzimologia , Bactérias/isolamento & purificação , Isópteros/microbiologia , Aerobiose , Anaerobiose , Animais , Aspergillus/classificação , Bactérias/classificação , Proteínas de Bactérias/metabolismo , Celulase/metabolismo , Técnicas de Cultura , DNA Bacteriano/análise , DNA Ribossômico/análise , Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Trato Gastrointestinal/microbiologia , Temperatura , alfa-Amilases/metabolismo
10.
Environ Entomol ; 42(5): 882-7, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-24331601

RESUMO

The complex microbial community living in the hindgut of lower termites includes prokaryotes, flagellates, yeasts, and filamentous fungi. Many microorganisms are found in the termite gut, but only a few are thought to be involved in symbiotic association to participate in cellulose digestion. Proteomics provides analyses from both taxonomical and functional perspectives. We aimed to identify symbiont diversity in the gut of Reticulitermes santonensis (Feytaud), via complementary electrospray ionization associated to ion trap tandem mass spectrometry (LC-MS/MS) and two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry analysis. One specific challenge to the study of lower termites is the relatively few data available on abundant symbiotic flagellates. Analysis based on LC-MS/MS revealed few protein families showing assignments to eukaryotes and the taxonomic origin of highly represented actins could not be established. Tubulins proved to be the most suitable protein family with which to identify flagellate populations from hindgut samples using LC-MS/MS, compared with other protein families, although this method targeted few prokaryotes in our assay. Similarly, two-dimensional gel electrophoresis associated to matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry did not succeed in identifying flagellate populations, but did permit the identification of most of the prokaryotic components of the symbiotic system. Finally, fungi and yeasts were identified by both methods. Owing to the lack of sequenced genes in flagellates, targeting tubulins for LC-MS/MS could allow fingerprints of flagellate populations to be established. Experimental and technical improvements might increase the efficiency of identification of prokaryotic populations in the near future, based on metaproteomic development.


Assuntos
Isópteros/microbiologia , Isópteros/fisiologia , Proteoma , Simbiose , Animais , Eucariotos/genética , Eucariotos/isolamento & purificação , França , Proteínas Fúngicas/genética , Fungos/genética , Fungos/isolamento & purificação , Trato Gastrointestinal/microbiologia , Proteínas de Protozoários/genética , Espectrometria de Massas por Ionização por Electrospray , Leveduras/genética , Leveduras/isolamento & purificação
11.
Springerplus ; 2: 410, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24024096

RESUMO

A novel serine protease gene, SBcas3.3, was identified by functional screening of a forest-soil metagenomic library on agar plates supplemented with AZCL-casein. Overproduction in Escherichia coli revealed that the enzyme is produced as a 770-amino-acid precursor which is processed to a mature protease of ~55 kDa. The latter was purified by affinity chromatography for characterization with the azocasein substrate. The enzyme proved to be an alkaline protease showing maximal activity between pH 9 and 10 and at 50°C. Treatment with the chelating agent ethylenediaminetetraacetic acid irreversibly denatured the protease, whose stability was found to depend strictly on calcium ions. The enzyme appeared relatively resistant to denaturing and reducing agents, and its activity was enhanced in the presence of 10 ml/l nonionic detergent (Tween 20, Tween 80, or Triton X-100). Moreover, SBcas3.3 displayed oxidant stability, a feature particularly sought in the detergent and bleaching industries. SBcas3.3 was activated by hydrogen peroxide at concentrations up to 10 g/l and it still retained 30% of activity in 50 g/l H2O2.

12.
Appl Biochem Biotechnol ; 171(1): 225-45, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23828225

RESUMO

The aim of this work was the isolation of xylanolytic microorganisms from the digestive tract of the termite Reticulitermes santonensis. The reducing sugars released after the hydrolysis of xylans can be further fermented to provide bioethanol. A xylanolytic strain of Bacillus subtilis was isolated from the hindgut of the termite and displayed amylase and xylanase activities. The bacterium was grown on media containing agricultural residues: wheat bran, wheat distiller's grains, and rapeseed oil cake. Wheat bran led to the highest induction of xylanase activity, although the development of the strain was less fast than in the other media. It was possible to reach maximal xylanase activities of 44.3, 33.5, and 29.1 I.U./ml in the media containing wheat bran, wheat distiller's grains, and rapeseed oil cake, respectively. Mass spectrometry identified a wide range of xylose oligomers, highlighting an endoxylanase activity. The enzyme was stable up to 45 °C and displayed an optimal pH close to 8.


Assuntos
Bacillus subtilis/crescimento & desenvolvimento , Bacillus subtilis/isolamento & purificação , Bacillus subtilis/metabolismo , Técnicas de Cultura , Intestinos/microbiologia , Isópteros/microbiologia , Xilanos/metabolismo , Sequência de Aminoácidos , Animais , Bacillus subtilis/genética , Betula/química , Endo-1,4-beta-Xilanases/biossíntese , Endo-1,4-beta-Xilanases/química , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Temperatura
13.
J Microbiol Biotechnol ; 23(6): 850-5, 2013 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-23676924

RESUMO

Finding new antimicrobial activities by functional metagenomics has been shown to depend on the heterologous host used to express the foreign DNA. Therefore, efforts are devoted to developing new tools for constructing metagenomic libraries in shuttle vectors replicatable in phylogenetically distinct hosts. Here we evaluated the use of the Escherichia coli-Bacillus subtilis shuttle vector pHT01 to construct a forest-soil metagenomic library. This library was screened in both hosts for antimicrobial activities against four opportunistic bacteria: Proteus vulgaris, Bacillus cereus, Staphylococcus epidermidis, and Micrococcus luteus. A new antibacterial activity against B. cereus was found upon screening in B. subtilis. The new antimicrobial agent, sensitive to proteinase K, was not active when the corresponding DNA fragment was expressed in E. coli. Our results validate the use of pHT01 as a shuttle vector and B. subtilis as a host to isolate new activities by functional metagenomics.


Assuntos
Anti-Infecciosos/metabolismo , Bacillus subtilis/genética , Biblioteca Gênica , Metagenoma , Microbiologia do Solo , Bacillus cereus/efeitos dos fármacos , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Micrococcus luteus/efeitos dos fármacos , Dados de Sequência Molecular , Proteus vulgaris/efeitos dos fármacos , Análise de Sequência de DNA , Staphylococcus epidermidis/efeitos dos fármacos
14.
Protein J ; 32(2): 126-30, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23385445

RESUMO

The yeast two-hybrid system is a powerful tool for detecting binary protein interactions, widely used in large-scale interactome mapping. We modified two yeast strains commonly used in yeast two-hybrid experiments by integrating into their genomes a new reporter gene encoding the enhanced yellow fluorescent protein eYFP. The suitability of this reporter gene for interaction screening was evaluated by fluorescence microscopy and fluorescence-activated cell sorting analysis. The gene shows good potential as a two-hybrid reporter gene for detecting strong interactions. Gal4 transcriptional activation gives rise to sufficient fluorescence for detection with a flow cytometer, but the eYFP reporter is not sensitive enough for detecting weak or moderate interactions. This study highlights the advantages of a fluorescent reporter gene in yeast two-hybrid screening.


Assuntos
Proteínas de Bactérias/metabolismo , Genes Reporter , Proteínas Luminescentes/metabolismo , Saccharomyces cerevisiae/metabolismo , Técnicas do Sistema de Duplo-Híbrido/instrumentação , Proteínas de Bactérias/genética , Engenharia Genética , Proteínas Luminescentes/genética , Saccharomyces cerevisiae/genética
15.
Plant Physiol Biochem ; 58: 245-52, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22858529

RESUMO

One mechanism used by plants to respond to infection is the production of antimicrobial peptides (AMPs). In addition to a role in defence, AMPs seem to have other biological functions. Furthermore, the number of cysteine-rich AMP-like peptides appears to have been underpredicted in plant genomes. Such peptides could be involved in plant defence and/or in other biological processes. Here we generated an interaction network between 15 AMPs/AMP-like peptides and ca. 8000 other Arabidopsis thaliana proteins (AtORFeome2.0) and found 53 putative novel interactions. These interactions involve five transcription factors, a subunit of the COP9 signalosome, a heat shock protein, a MAP kinase kinase, a thioredoxin and 4 uncharacterized proteins.


Assuntos
Anti-Infecciosos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Resistência à Doença , Peptídeos/metabolismo , Doenças das Plantas/microbiologia , Imunidade Vegetal , Complexo do Signalossomo COP9 , Cisteína/metabolismo , Genoma de Planta , Proteínas de Choque Térmico/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Complexos Multiproteicos/metabolismo , Peptídeo Hidrolases/metabolismo , Subunidades Proteicas , Tiorredoxinas/metabolismo , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
16.
Protein Expr Purif ; 83(2): 117-27, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22487213

RESUMO

Termites are world champions at digesting lignocellulosic compounds, thanks to cooperation between their own enzymes and exogenous enzymes from microorganisms. Prokaryotic cells are responsible for a large part of this lignocellulolytic activity. Bacterial enzyme activities have been demonstrated in the higher and the lower termite gut. From five clones of Gram-positive bacteria isolated and identified in a previous work, we constructed a genomic DNA library and performed functional screening for alpha-amylase, beta-glucosidase, and xylanase activities. One candidate, Xyl8B8, showed xylanase activity. Sequence analysis of the genomic insert revealed five complete ORFs on the cloned DNA (5746bp). Among the encoded proteins were a putative endo-1,4-beta-xylanase (XylB8) belonging to glycoside hydrolase family 11 (GH11). On the basis of sequence analyses, genomic DNA organization, and phylogenetic analysis, the insert was shown to come from an actinobacterium. The mature xylanase (mXylB8) was expressed in Escherichia coli and purified by affinity chromatography and detected by zymogram analysis after renaturing. It showed maximal xylanase activity in sodium acetate buffer, pH 5.0 at 55 °C. Its activity was increased by reducing agents and decreased by Cu(2+), some detergents, and chelating agents. Its substrate specificity appeared limited to xylan.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Isópteros/microbiologia , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Sequência de Bases , Cromatografia de Afinidade , Clonagem Molecular , Endo-1,4-beta-Xilanases/isolamento & purificação , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Trato Gastrointestinal/microbiologia , Glicosídeo Hidrolases , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Filogenia , Análise de Sequência de DNA , Xilanos/metabolismo
17.
Fungal Biol ; 115(11): 1093-9, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22036288

RESUMO

Several Ascomycetes fungi are commonly used in bio-industries and provide available industrial residues for lectin extraction to be valuable. A lectin from Penicillium chrysogenum, named PeCL, was purified from a fungal culture using gel-filtration chromatography column. PeCL was found to be a mannose-specific lectin by haemagglutination activity towards rabbit erythrocyte cells and was visualised on SDS-PAGE gel. Purified PeCL fraction was delivered via artificial diet to Myzus persicae aphid and was demonstrated to be aphicidal at 0.1 % with higher toxic efficiency than a known mannose-binding lectin Concanavalin A (ConA). A fast and efficient way to purify PeCL and a potential use in pest control is described.


Assuntos
Afídeos/efeitos dos fármacos , Proteínas Fúngicas/isolamento & purificação , Inseticidas/isolamento & purificação , Lectinas de Ligação a Manose/isolamento & purificação , Penicillium chrysogenum/química , Animais , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/toxicidade , Testes de Hemaglutinação , Inseticidas/química , Inseticidas/metabolismo , Inseticidas/toxicidade , Lectinas de Ligação a Manose/química , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/toxicidade , Penicillium chrysogenum/metabolismo , Coelhos
18.
Microbiol Res ; 166(8): 629-42, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-21324659

RESUMO

ß-Glucosidases are widely distributed in living organisms and play a major role in the degradation of wood, hydrolysing cellobiose or cello-oligosaccharides to glucose. Termites are among the rare animals capable of digesting wood, thanks to enzyme activities of their own and to enzymes produced by their gut microbiota. Many bacteria have been identified in the guts of lower termites, some of which possess cellulolytic or/and hemicellulolytic activity, required for digesting wood. Here, having isolated bacterial colonies from the gut of Reticulitermes santonensis, we constructed in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained and screened the library for clones displaying ß-glucosidase activity. This screen revealed 8 positive clones. Sequence analysis with the BLASTX program revealed putative enzymes belonging to three glycoside hydrolase families (GH1, GH3 and GH4). Agar-plate tests and enzymatic assays revealed differences between the GH1- and GH3-type enzymes (as regards substrate specificity and regulation) and a difference in substrate specificity within the GH3 group. The substrate specificities and characteristic activities of these enzymes suggest that they may intervene in the depolymerisation of cellulose and hemicellulose.


Assuntos
Biblioteca Gênica , Isópteros/microbiologia , Metagenoma , beta-Glucosidase/análise , beta-Glucosidase/genética , Ágar , Animais , Celulose/metabolismo , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , Escherichia coli/genética , Trato Gastrointestinal/microbiologia , Dados de Sequência Molecular , Polissacarídeos/metabolismo , Análise de Sequência de DNA , Especificidade por Substrato
19.
Biotechnol Lett ; 33(1): 123-9, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-20872164

RESUMO

The yeast proteins, Msb3p and Msb4p, are two Ypt/Rab-specific GTPase-activating proteins sharing redundant functions in exocytosis, organization of the actin cytoskeleton, and budding site selection. To see if Msb3p might play an additional, specific role, we first tested the sensitivities of msb3 and msb4 mutant strains to different drugs and then screened a genomic library for multicopy suppressors of msb3 sensitivity to CdCl(2) or to the calcium channel blocker diltiazem hydrochloride. Three genes (ADH1, RNT1, and SUI1) were found to suppress the CdCl(2) sensitivity of the msb3 strain and three others (YAP6, ZEO1, and SLM1) its diltiazem-HCl sensitivity. The results suggest a possible involvement of Msb3p in calcineurin-mediated signalling.


Assuntos
Proteínas Ativadoras de GTPase/deficiência , Proteínas Ativadoras de GTPase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Supressão Genética , Proteínas rab de Ligação ao GTP/metabolismo , Antifúngicos/farmacologia , Cloreto de Cádmio/farmacologia , Diltiazem/farmacologia , Genes Fúngicos , Genética Microbiana/métodos , Programas de Rastreamento/métodos , Testes de Sensibilidade Microbiana , Saccharomyces cerevisiae/efeitos dos fármacos
20.
FEMS Microbiol Lett ; 314(2): 147-57, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21114521

RESUMO

The gut of the termite Reticulitermes santonensis contains an interesting diversity of prokaryotic and eukaryotic microorganisms not found elsewhere. These microorganisms produce many enzyme-digesting lignocellulosic compounds, probably in cooperation with endogenous enzymes. Regarding cellulose and hemicellulose digestion in the termite gut, much remains to be learned about the relative contributions of termite enzymes and enzymes produced by different microorganisms. Here we grew bacterial colonies from termite gut suspensions, identifying 11 of them after PCR amplification of their 16S rRNA genes. After constructing in Escherichia coli a genomic DNA library corresponding to all of the colonies obtained, we performed functional screening for α-amylase, xylanase, ß-glucosidase, and endoglucanase activities. This screen revealed a clone producing ß-glucosidase activity. Sequence analysis showed that the cloned genomic DNA fragment contained three complete ORFs (bglG, bglF, and bglB) organized in a putative bgl operon. The new ß-glucosidase (BglB), identified with its regulators BglG and BglF, belongs to glycoside hydrolase family 1. The new ß-glucosidase was expressed in E. coli and purified by affinity chromatography. The purified enzyme shows maximal activity at pH 6.0 and 40 °C. It also displays ß-xylosidase activity.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/metabolismo , Isópteros/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Sequência de Aminoácidos , Animais , Bactérias/classificação , Bactérias/isolamento & purificação , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Escherichia coli/genética , Trato Gastrointestinal/microbiologia , Biblioteca Gênica , Dados de Sequência Molecular , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Xilosidases/genética , beta-Glucosidase/genética
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