RESUMO
Twelve Bacillus thuringiensis (Bt) strains, isolated from larvae and soil samples in Argentina, were molecularly and phenotypically characterized and their insecticidal activities against Spodoptera frugiperda and Peridroma saucia were determined. One isolate--Bt RT--produced more than 93% mortality on first instar larvae of both species, which was higher than that produced by the reference strain Bt 4D1. Bt RT carried a different cry gene profile than Bt 4D1. Scanning electron microscopy showed the presence of bipyramidal and cuboidal crystals. Phenotypic characterization revealed lytic enzymes that could contribute to Bt pathogenicity.
Assuntos
Bacillus thuringiensis/isolamento & purificação , Bacillus thuringiensis/patogenicidade , Proteínas de Bactérias/toxicidade , Endotoxinas/toxicidade , Infecções por Bactérias Gram-Positivas/veterinária , Proteínas Hemolisinas/toxicidade , Lepidópteros/efeitos dos fármacos , Lepidópteros/microbiologia , Animais , Argentina , Bacillus thuringiensis/ultraestrutura , Toxinas de Bacillus thuringiensis , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Endotoxinas/metabolismo , Infecções por Bactérias Gram-Positivas/microbiologia , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/ultraestrutura , Larva/microbiologia , Microscopia Eletrônica de Varredura , Microbiologia do Solo , Spodoptera/efeitos dos fármacos , Spodoptera/microbiologia , Análise de SobrevidaRESUMO
Fourteen strains of Bacillus thuringiensis collected from both larvae showing disease symptoms and soil samples in northwest Argentina were characterized by insecticidal activity against Spodoptera frugiperda. First instar larvae and protein profile SDS-PAGE analysis of whole cell proteins not only allowed the differentiation of native Bacillus thuringiensis but also revealed the possibility of applying protein profile analysis in classification of toxicity patterns. Cluster analysis showed that there were two main groups. Interestingly, one of them only contained the most pathogenic native strains. The biomass-bound protease activity of native pathogenic isolates and the reference strain Bt 4D1 is also reported.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/farmacologia , Endotoxinas/farmacologia , Proteínas Hemolisinas/farmacologia , Análise Numérica Assistida por Computador , Controle Biológico de Vetores , Spodoptera/efeitos dos fármacos , Animais , Bacillus thuringiensis/enzimologia , Bacillus thuringiensis/isolamento & purificação , Toxinas de Bacillus thuringiensis , Biomassa , Eletroforese em Gel de Poliacrilamida , Larva/efeitos dos fármacos , Larva/microbiologia , Dados de Sequência Molecular , Peptídeo Hidrolases/metabolismo , Filogenia , Microbiologia do SoloRESUMO
New thermophilic and organic-solvent-tolerant Bacillus licheniformis S-86 strain is able to produce two active and solvent-stable esterases. Production of type I and II esterases was substantially enhanced when oils and surfactants were supplied as carbon sources. Grape oil (0.1% v/v) and Tween 20 to 60 (0.1% v/v) had enhanced enzyme production between 1.6- and 2.2-folds. Type II esterase was purified to homogeneity in a five-step procedure. This esterase was purified 76.7-fold with a specific activity of 135 U mg(-1). Molecular mass of the enzyme was estimated to be 38.4 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Type II esterase was active mostly on esters with short acyl chains, which allowed to classify the enzyme as a carboxylesterase with a K (m) of 80.2 mmol l(-1) and a V (max) of 256.4 micromol min(-1) mg(-1) for p-nitrophenyl acetate. Also, B. licheniformis S-86 type II esterase displayed activity in presence of water-miscible organic solvents at 50% concentration and stability after 1-h incubation.
Assuntos
Bacillus/enzimologia , Hidrolases de Éster Carboxílico/biossíntese , Bacillus/efeitos dos fármacos , Hidrolases de Éster Carboxílico/efeitos dos fármacos , Hidrolases de Éster Carboxílico/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Estabilidade Enzimática , Cinética , Óleos de Plantas/farmacologia , Polissorbatos/farmacologia , Solventes/farmacologia , Especificidade por Substrato , Tensoativos/farmacologia , VitisRESUMO
A constitutive level of a mycelium-bound lipolytic activity from Aspergillus niger MYA 135 was strongly increased by 97% in medium supplemented with 2% olive oil. The constitutive lipase showed an optimal activity in the pH range of 3.0-6.5, while the mycelium-bound lipase activity produced in the presence of olive oil had two pH optima at pH 4 and 7. Interestingly, both lipolytic sources were cold-active showing high catalytic activities in the temperature range of 4-8 degrees C. These mycelium-bound lipase activities were also very stable in reaction mixtures containing methanol and ethanol. In fact, the constitutive lipase maintained almost 100% of its activity after exposure by 1 h at 37 degrees C in ethanol. A simple methodology to evaluate suitable transesterification activities in organic solvents was also reported.
Assuntos
Aspergillus niger/enzimologia , Microbiologia Industrial/métodos , Lipase/metabolismo , Álcoois/metabolismo , Catálise , Estabilidade Enzimática , Fermentação , Concentração de Íons de Hidrogênio , Micélio/enzimologia , Azeite de Oliva , Óleos de Plantas/farmacologia , Solventes , TemperaturaRESUMO
The BhMIR32 xyn11A gene, encoding an extracellular endoxylanase of potential interest in bio-bleaching applications, was amplified from Bacillus halodurans MIR32 genomic DNA. The protein encoded is an endo-1,4-beta-xylanase belonging to family 11 of glycosyl hydrolases. Its nucleotide sequence was analysed and the mature peptide was subcloned into pET22b(+) expression vector. The enzyme was over-expressed in a high density Escherichia coli culture as a soluble and active protein, and purified in a single step by immobilised metal ion affinity chromatography with a specific activity of 3073 IU mg-1.
Assuntos
Bacillus/genética , DNA Bacteriano/genética , Endo-1,4-beta-Xilanases/genética , Bacillus/enzimologia , Proteínas de Bactérias/genética , Sequência de Bases , Sítios de Ligação/genética , Clonagem Molecular , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Dados de Sequência Molecular , Regiões Promotoras Genéticas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Análise de Sequência de DNARESUMO
The flavonoids, constituting one of the most numerous and widespread groups of natural plant constituents, are important to humans not only because they contribute to plant colors but also because many members are physiologically active. These low-molecular-weight substances, found in all vascular plants, are phenylbenzopyrones. Over 4000 structures have been identified in plant sources, and they are categorized into several groups. Primarily recognized as pigments responsible for the autumnal burst of hues and the many shades of yellow, orange, and red in flowers and food, the flavonoids are found in fruits, vegetables, nuts, seeds, stems, flowers, and leaves as well as tea and wine and are important constituents of the human diet. They are prominent components of citrus fruits and other food sources. Flavonols (quercetin, myricetin, and kaempferol) and flavones (apigenin and luteolin) are the most common phenolics in plant-based foods. Quercetin is also a predominant component of onions, apples, and berries. Such flavanones as naringin are typically present in citrus fruit, and flavanols, particularly catechin, are present as catechin gallate in such beverages as green or black tea and wine. Some major sources of flavonoids are outlined in Table 1. The daily intake of flavonoids in humans has been estimated to be approx 25 mg/d, a quantity that could provide pharmacologically significant concentrations in body fluids and tissues, assuming good absorption from the gastrointestinal tract. Biological activity of flavonoids was first suggested by Szent-Gÿorgyi 1938, who reported that citrus peel flavonoids were effective in preventing the capillary bleeding and fragility associated with scurvy. The broad spectrum of biological activity within the group and the multiplicity of actions displayed by a certain individual members make the flavonoids one of the most promising classes of biologically active compounds.
Assuntos
Anti-Infecciosos/isolamento & purificação , Asteraceae/química , Flavonoides/isolamento & purificação , Anti-Infecciosos/química , Argentina , Cromatografia em Papel/métodos , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Flavonoides/química , Indicadores e Reagentes , Testes de Sensibilidade Microbiana/métodos , Modelos Moleculares , Estrutura Molecular , Fitoterapia , SolventesRESUMO
Bacillus sp. MIR32 has been isolated using xylan as the only carbon source, and one of its xylanolytic enzymes has been extensively studied. Biochemical analysis first related this strain to Bacillus amyloliquefaciens, but further studies based on a comparison of 16S rDNA sequences, G+C content, and DNA-DNA hybridization showed that strain MIR32 should be classified as a member of the species Bacillus halodurans. This change is also supported by the typical phenotype observed and by the results of PCR amplification directed toward spacers in rDNA and tDNA genes, which were assayed and compared with those of B. halodurans DSM 497(T). Although among alkaliphilic bacilli competence development has not been experimentally demonstrated, in this work both B. halodurans MIR32 and DSM 497(T) were transformed according to a simple procedure developed in our laboratory, reaching 10(2)-10(3) stable transformants per microgram of plasmid DNA.