RESUMO
DNA electrophoresis is a fundamental technique in molecular biology that allows the separation of DNA molecules up to ~50 Kbp. Pulsed-field gel electrophoresis [PFGE] is a variation of the conventional DNA electrophoresis technique that allows the separation of very large DNA molecules up to ~10 Mbp. PFGE equipment is very expensive and it becomes an access barrier to many laboratories. Also, just a few privative designs of the equipment are available and it becomes difficult for the community to improve or customize their functioning. Here, we provide an open source PFGE equipment capable of the separation of DNA molecules up to, at least, ~2 Mbp and at low cost: USD$850, about 3% of the price of typical commercial equipment.
RESUMO
Alginate lyases (endo and exo-lyases) are required for the degradation of alginate into its constituting monomers. Efficient bioethanol production and extraction of bioactives from brown algae requires intensive use of these enzymes. Nonetheless, there are few commercial alginate lyase preparations, and their costs make them unsuitable for large scale experiments. A recombinant expression protocol has been developed in this study for producing seven endo-lyases and three exo-lyases as soluble and highly active preparations. Saccharification of alginate using 21 different endo/exo-lyase combinations shows that there is complementary enzymatic activity between some of the endo/exo pairs. This is probably due to favorable matching of their substrate biases for the different glycosidic bonds in the alginate molecule. Therefore, selection of enzymes for the best saccharification results for a given biomass should be based on screens comprising both types of lyases. Additionally, different incubation temperatures, enzyme load ratios, and enzyme loading strategies were assessed using the best four enzyme combinations for treating Macrocystis pyrifera biomass. It was shown that 30°C with a 1:3 endo/exo loading ratio was suitable for all four combinations. Moreover, simultaneous loading of endo-and exo-lyases at the beginning of the reaction allowed maximum alginate saccharification in half the time than when the exo-lyases were added sequentially.
Assuntos
Alginatos/metabolismo , Microbiologia Industrial/métodos , Polissacarídeo-Liases/biossíntese , Polissacarídeo-Liases/metabolismo , Alga Marinha/química , Biocombustíveis , Clonagem Molecular , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Polissacarídeo-Liases/classificação , Polissacarídeo-Liases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Alga Marinha/metabolismo , TemperaturaRESUMO
Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.
Assuntos
Basidiomycota/enzimologia , Celulases/metabolismo , Polissacarídeos Fúngicos/metabolismo , Proteínas Fúngicas/metabolismo , Lignina/metabolismo , Oxigenases de Função Mista/metabolismo , Xilosidases/metabolismo , Basidiomycota/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Hidrólise , Oxigenases de Função Mista/química , Oxigenases de Função Mista/genética , Xilanos/metabolismo , Xilosidases/química , Xilosidases/genéticaRESUMO
Total cDNA isolated from cellulolytic fungi cultured in cellulose was examined for the presence of sequences encoding for endoglucanases. Novel sequences encoding for glycoside hydrolases (GHs) were identified in Fusarium oxysporum, Ganoderma applanatum and Trametes versicolor. The cDNA encoding for partial sequences of GH family 61 cellulases from F. oxysporum and G. applanatum shares 58 and 68% identity with endoglucanases from Glomerella graminicola and Laccaria bicolor, respectively. A new GH family 5 endoglucanase from T. versicolor was also identified. The cDNA encoding for the mature protein was completely sequenced. This enzyme shares 96% identity with Trametes hirsuta endoglucanase and 22% with Trichoderma reesei endoglucanase II (EGII). The enzyme, named TvEG, has N-terminal family 1 carbohydrate binding module (CBM1). The full length cDNA was cloned into the pPICZαB vector and expressed as an active, extracellular enzyme in the methylotrophic yeast Pichia pastoris. Preliminary studies suggest that T. versicolor could be useful for lignocellulose degradation.
Assuntos
Celulases/genética , Proteínas Fúngicas/genética , Trametes/enzimologia , Trametes/genética , Sequência de Aminoácidos , Sequência de Bases , Celulases/química , Celulases/metabolismo , Clonagem Molecular , DNA Fúngico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Expressão Gênica , Genes Fúngicos , Dados de Sequência Molecular , Filogenia , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de AminoácidosRESUMO
El Síndrome Premenstrual fue descrito ya por Hipócrates y es definido como la aparición cíclica de síntomas que pueden alterar la vida de la mujer y que aparecen en forma predecible en relación a los días previos a la regla. El Síndrome Premenstrual puede exagerar algunas patologías médicas y neuropsiquiátricas, en especial estas últimas cuando nos enfrentamos al Trastorno Disfórico Premenstrual, y debemos estar atentos a su diagnóstico...
Assuntos
Humanos , Dismenorreia/diagnóstico , Qualidade de Vida , Síndrome Pré-Menstrual/complicaçõesRESUMO
A set of poly(propylene) composites containing different amounts of copper nanoparticles (CNP) were prepared by the melt mixed method and their antimicrobial behavior was quantitatively studied. The time needed to reduce the bacteria to 50% dropped to half with only 1 v/v % of CNP, compared to the polymer without CNP. After 4 h, this composite killed more than 99.9% of the bacteria. The biocide kinetics can be controlled by the nanofiller content; composites with CNP concentrations higher than 10 v/v % eliminated 99% of the bacteria in less than 2 h. X-ray photoelectron spectroscopy did not detect CNP at the surface, therefore the biocide behavior was attributed to copper in the bulk of the composite.
RESUMO
Enzymatic methods provide a convenient alternative for overcoming technical disadvantages of mechanical disruption. Protocols for protein extraction from bacteria and Saccharomyces cerevisiae using lytic enzymes are presented in this chapter. Adaptation of the yeast protocol to a microtiter plate format makes this protocol amenable for proteomic applications and high-throughput screening of libraries expressing genetic variants in yeast. This methodology can also be applied to bacteria.
Assuntos
Bactérias/química , Proteínas de Bactérias/isolamento & purificação , Enzimas/química , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Saccharomyces cerevisiae/química , Proteínas de Bactérias/química , Proteínas de Saccharomyces cerevisiae/químicaRESUMO
The increased demand for enzymes with new properties makes indispensable the development of easy and rapid strategies to obtain complete genes of new enzymes. Here a strategy is described which includes screening by PCR of new subtilases mediated by Consensus-Degenerate Hybrid Oligonucleotide Primers (CODEHOP) and an improved genome walking method to obtain the complete sequence of the identified genes. Existing methods of genome walking have many limitations, which make them inefficient and time consuming. We have developed an improved genome walking method with novel advances to get a simple, rapid and more efficient procedure based on cassette-ligation. Improvements consist basically in the possibility of a genomic DNA digestion with any restriction enzyme, blunting and 3' adenylation of digested DNA by Taq DNA polymerase to avoid self-circularization, followed by TA ligation of the adenine 3' overhanging end to the same unphosphorylated oligo-cassette. The efficiency of the genome walking method was demonstrated by finding the unknown ends of all gene fragments tested, previously obtained by CODEHOP-mediated PCR, including three subtilases (P4, P6 and P7), one xylanase and one lipase, from different strains of Antarctic marine bacteria.
Assuntos
Bactérias/enzimologia , Bactérias/genética , Passeio de Cromossomo/métodos , Genes Bacterianos/genética , Genoma Bacteriano/genética , Água do Mar/microbiologia , Microbiologia da Água , Regiões Antárticas , Sequência de Bases , Clonagem Molecular , Sequência Consenso , Enzimas de Restrição do DNA , Biblioteca Gênica , Hidrólise , Filogenia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Análise de Sequência de Proteína , Solubilidade , Subtilisina/genéticaRESUMO
Cell wall lytic enzymes are valuable tools for the biotechnologist, with many applications in medicine, the food industry, and agriculture, and for recovering of intracellular products from yeast or bacteria. The diversity of potential applications has conducted to the development of lytic enzyme systems with specific characteristics, suitable for satisfying the requirements of each particular application. Since the first time the lytic enzyme of excellence, lysozyme, was discovered, many investigations have contributed to the understanding of the action mechanisms and other basic aspects of these interesting enzymes. Today, recombinant production and protein engineering have improved and expanded the area of potential applications. In this review, some of the recent advances in specific enzyme systems for bacteria and yeast cells rupture and other applications are examined. Emphasis is focused in biotechnological aspects of these enzymes.
Assuntos
Bactérias/citologia , Bactérias/metabolismo , Enzimas/metabolismo , Leveduras/citologia , Leveduras/metabolismo , BacterióliseRESUMO
BGLII is a bacterial endoglucanase that hydrolyzes the beta-1,3-glucan present in yeast cell walls, resulting in lysis of Saccharomyces cerevisiae. As a result of this property, BGLII is considered a potential tool for downstream processing and recovery of biotechnological products produced in yeast. Here we describe the improvement of the yeast lytic activity of BGLII, achieved by a directed evolution approach involving random mutagenesis and screening for variants with improved catalytic activity, combined with site-directed mutagenesis. A BGLII variant having three times the wild-type hydrolytic activity on laminarin was identified. The purified enzyme also exhibited higher lytic activity on yeast cells. Mutations causing the improvements are located very close to each other in the amino acid sequence, suggesting that the region should be considered as a target for further improvements of the glucanase activity. These results demonstrate the feasibility of molecular evolution methods for the improvement of the BGLII hydrolytic activity, and open a window for further improvement of this or other properties in glycosyl hydrolases in general.