RESUMO
Biodiesels are mostly produced from lipid transesterification of vegetable oils, including those from soybean, jatropha, palm, rapeseed, sunflower, and others. Unfortunately, transesterification of oil produces various unwanted side products, including steryl glucosides (SG), which precipitate and need to be removed to avoid clogging of filters and engine failures. So far, efficient and cost-effective methods to remove SGs from biodiesel are not available. Here we describe for the first time the identification, characterization and heterologous production of an enzyme capable of hydrolyzing SGs. A synthetic codon-optimized version of the lacS gene from Sulfolobus solfataricus was efficiently expressed and purified from Escherichia coli, and used to treat soybean derived biodiesel containing 100 ppm of SGs. After optimizing different variables, we found that at pH 5.5 and 87 °C, and in the presence of 0.9 % of the emulsifier polyglycerol polyricinoleate, 81 % of the total amount of SGs present in biodiesel were hydrolyzed by the enzyme. This remarkable reduction in SGs suggests a path for the removal of these contaminants from biodiesel on industrial scale using an environmentally friendly enzymatic process.
Assuntos
Biocombustíveis , Colestenos/metabolismo , Hidrolases/metabolismo , Sulfolobus solfataricus/enzimologia , DNA Arqueal/química , DNA Arqueal/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Concentração de Íons de Hidrogênio , Hidrolases/genética , Hidrolases/isolamento & purificação , Hidrólise , Dados de Sequência Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Análise de Sequência de DNA , Óleo de Soja , TemperaturaRESUMO
A structure-activity relationship around the amine group of the ambruticin VS series has been developed for antifungal activity. It was shown that the amine can be alkylated through reductive amination without loss of potency. However, if it is converted into either an amide, carbamate, or urea, a significant loss of potency is observed. Of the alkyl amines, small nonpolar groups are optimal for both potency and oral bioavailability. As a result of this study, one compound (KOS-2079) was taken into an animal efficacy model with success.
Assuntos
Aminas/química , Antifúngicos/farmacologia , Coccidioides/efeitos dos fármacos , Alquilação , Aminação , Animais , Antifúngicos/síntese química , Antifúngicos/química , Disponibilidade Biológica , Desenho de Fármacos , Camundongos , Testes de Sensibilidade Microbiana , Conformação Molecular , Piranos/síntese química , Piranos/química , Piranos/farmacologia , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
The polyketide ambruticin is an attractive candidate for drug development as an antifungal agent, but its mechanism of action has not yet been elucidated. Here we present evidence that ambruticin exerts its effect by targeting HOG, the osmotic stress control pathway, through Hik1, a group III histidine kinase.
Assuntos
Antifúngicos/farmacologia , Fungos/efeitos dos fármacos , Glicerol/farmacologia , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais/efeitos dos fármacos , Western Blotting , Testes de Sensibilidade Microbiana , Concentração Osmolar , Fosforilação , Piranos/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Leveduras/efeitos dos fármacosRESUMO
Genetic manipulation of antibiotic producers, such as Streptomyces species, is a rational approach to improve the properties of biologically active molecules. However, this can be a slow and sometimes problematic process. Red/ET recombination in an Escherichia coli host has permitted rapid and more versatile engineering of geldanamycin biosynthetic genes in a complementation plasmid, which can then be readily transferred into the Streptomyces host from which the corresponding wild type gene(s) has been removed. With this rapid Red/ET recombination and gene complementation approach, efficient gene disruptions and gene replacements in the geldanamycin biosynthetic gene cluster have been successfully achieved. As an example, we describe here the creation of a ketoreductase 6 null mutation in an E. coli high-copy-number plasmid carrying gdmA2A3 from Streptomyces hygroscopicus NRRL3602 and the subsequent complementation of a gdmA2A3 deletion host with this plasmid to generate a novel geldanamycin analog.
Assuntos
Proteínas de Bactérias/genética , Teste de Complementação Genética , Engenharia Genética/métodos , Quinonas/metabolismo , Recombinação Genética , Streptomyces/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Bacteriófago lambda/enzimologia , Bacteriófago lambda/genética , Benzoquinonas , Conjugação Genética , DNA Bacteriano/análise , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Deleção de Genes , Lactamas Macrocíclicas , Plasmídeos , Quinonas/química , Streptomyces/metabolismo , Fatores de TempoRESUMO
A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.
