RESUMEN
Protein profiles of the basidiomycete Trametes hirsuta grown on standard medium without laccase as an inducer and on medium supplemented with CuSO4 were analyzed using a differential proteomics approach. Protocols developed for isolation and purification of extracellular and intracellular proteins of the mycelium allowed us to show extensive extraction of protein components. Simultaneously, components hampering two-dimensional electrophoresis (pigments, low molecular mass metabolites) were removed from the samples, and high-resolution protein maps were obtained. Analysis of the basidiomycete secretomes revealed qualitative changes in the protein profile: the addition of CuSO4 as an inducer resulted in increase in the produced laccase isoforms and/or isozymes from 7 to 11, whereas its pI range change exceeded 2 units. The number of separated intracellular protein components was 552 and 502 for the control medium and medium with the inducer, respectively. Comparative analysis of the protein maps revealed five regions with the most pronounced differences in the protein profiles. The proteins of interest were identified using MALDI-TOF/TOF mass spectrometry with subsequent peptide fingerprinting. Some intracellular proteins (ß-subunits of ATP synthase, molecular chaperones, chaperone activator) upregulated during the growth on the inducer-containing medium were identified. These proteins are supposed to be involved in the regulation of laccase biosynthesis during folding and secretion of the enzyme.
Asunto(s)
Medios de Cultivo/metabolismo , Proteínas Fúngicas/química , Proteómica , Trametes/metabolismo , Medios de Cultivo/química , Electroforesis en Gel Bidimensional , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Trametes/química , Trametes/genética , Trametes/crecimiento & desarrolloRESUMEN
A comparative analysis of transcripts from the basidiomycota T. hirsuta grown with and without an inducer of the laccase biosynthesis was carried out. Methods of subtraction hybridization and massive parallel sequencing were used for this purpose. Unique transcripts encoded by genes that have a relatively high level of expression and belong to different gene ontology categories were identified. Also, a large number of transcripts were found to encode for predicted proteins, as well as noncoding transcripts. The latter may represent regulatory RNA molecules. Transcripts that increase their abundance when the laccase synthesis is induced are selected as gene-candidates involved in the laccase biosynthetic pathway.
Asunto(s)
Proteínas Fúngicas/genética , Lacasa/biosíntesis , Trametes/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Lacasa/genética , Trametes/crecimiento & desarrollo , Trametes/metabolismoRESUMEN
A heterologous protein expression in the fungus Penicillium canescens is described for the first time. The fungal strains producing Trametes hirsuta laccase under control of a highly efficient promoter of the P. canescens gene bgaS has been constructed. These strains efficiently transcribe the T. hirsuta 072 laccase gene with a correct intron splicing. Activity of the secreted heterologous laccase in the culture liquid reaches 3 U/ml, accounting for 98% of the total laccase activity, which demonstrates a high efficiency ofheterologous secretion. The synthesized P. canescens laccase has the same molecular weight as the enzyme produced by T. hirsuta 072.
Asunto(s)
Proteínas Fúngicas/biosíntesis , Expresión Génica , Penicillium/crecimiento & desarrollo , Proteínas Recombinantes/biosíntesis , Trametes/enzimología , Proteínas Fúngicas/genética , Lacasa , Penicillium/genética , Regiones Promotoras Genéticas/genética , Proteínas Recombinantes/genética , Trametes/genéticaRESUMEN
Gene egl2 of secreted endo-(1-4)-beta-glucanase of glycosyl hydrolase family 5 of the mycelial fungus Penicillium canescens was cloned. The gene was expressed in P. canescens under control of a strong promoter of the bgaS gene encoding beta-galactosidase of P. canescens, and endoglucanase producing strains were obtained. Chromatographically purified recombinant 48 kDa protein had pH and temperature optima 3.4 and 60 degrees C, respectively, exhibited specific activity of 33 IU, and had K(m) and V(max) in CM-cellulose hydrolysis of 10.28 g/liter and 0.26 micromol/sec per mg, respectively.
Asunto(s)
Celulasa/genética , Celulasa/metabolismo , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Penicillium/enzimología , Secuencia de Aminoácidos , Secuencia de Bases , Rastreo Diferencial de Calorimetría , Carboximetilcelulosa de Sodio/metabolismo , Celulasa/biosíntesis , Celulasa/aislamiento & purificación , Estabilidad de Enzimas , Escherichia coli/genética , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Galactosidasas/genética , Galactosidasas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Cinética , Datos de Secuencia Molecular , Nitrato-Reductasa/genética , Nitrato-Reductasa/metabolismo , Señales de Clasificación de Proteína , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Transformación BacterianaRESUMEN
The gene egl3 of the filamentous fungus Penicillium canescens endo-1,4-beta-glucanase, belonging to family 12 glycosyl hydrolases, was cloned and sequenced. The gene was expressed in P. canescens under the control of the strong promoter of gene bgaS, coding for beta-galactosidase of this fungus, and efficient endoglucanase producer strains were obtained. The recombinant protein was isolated from the culture liquid of the producer strain EGL3-13 and purified to homogeneity; its specific activity was 31.7 IU; molecular weight, 26 kDa; and pH and temperature optimums, 3.2 and 54 degrees C, respectively. The Km and Vm values for CMC hydrolysis were determined; they amounted to 17.1 g/1 and 0.31 microM/(mg s), respectively.
Asunto(s)
Celulasa/biosíntesis , Celulasa/química , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Penicillium/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Celulasa/genética , Celulasa/aislamiento & purificación , Clonación Molecular/métodos , Proteínas Fúngicas/aislamiento & purificación , Expresión Génica , Calor , Concentración de Iones de Hidrógeno , Cinética , Penicillium/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
The goal of this work was to suggest a method for improving the efficiency of ECG analysis in measurements of the electrical cardiac activity. The suggested method is based on a solution of the inverse ECG problem. The algorithm for measuring the electrical cardiac activity includes storage of ECG signals from various sources in a database (the body and heart geometry are taken into account), determination of electrically active areas, and determination of the electrical activity at each point of the heart model surface. Test ECG signals available at a free-access web site were used to verify the results of monitoring of the electrical cardiac activity.
