Purification and characterization of a keratinolytic serine protease from Purpureocillium lilacinum LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.Fil: Cavello, Ivana Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Hours, Roque Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Rojas, Natalia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Cavalitto, Sebastian Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); Argentin

Purification and characterization of a novel alkaline α-L-rhamnosidase produced by Acrostalagmus luteo albus

Rhamnosidases are enzymes that catalyze the hydrolysis of terminal non reducing L-rhamnose for the bioconversion of natural or synthetic rhamnosides. They are of great significance in the current biotechnological area with applications in food and pharmaceutical industrial processes. In this study we isolated and characterized a novel alkaline rhamnosidase from Acrostalagmus luteo albus, an alkali tolerant soil fungus from Argentina. We also present here an efficient, simple, and inexpensive method for purifying the A. luteo albus rhamnosidase and describe the characteristics of the purified enzyme. In presence of rhamnose as sole carbon source, this fungus produces a rhamnosidase of 109 kDa molecular weight and a pI value of 4.6 determined by SDS-PAGE and analytical isoelectric focusing, respectively. This enzyme was purified to homogeneity by chromatographic and electroforetic techniques. Using p-nitrofenil--L rhamnopiranoside as substrate, the enzyme activity shows pH and temperature optima of 8.0 and 55 ºC, respectively. The enzyme exhibited Michaelis-Menten kinetics with KM and Vmax values of 3.38 mmol.l-1 and 68.5 mmol.l-1.min-1. Divalent cations such as Ca+2, Mg+2, Mn+2, and Co+2 or reducing agents such as -mercaptoethanol and dithiothreitol showed no effect over enzyme activity, whereas this was completely inhibited by Zn+2 at a concentration of 0.2 mM. This enzyme showed the capability to hydrolyze some natural rhamnoglucosides such as hesperidin, naringin and quercitrin under alkaline conditions. On the basis of these results, and mainly due to the high activity of the A. luteo albus rhamnosidase under alkaline conditions, this enzyme should be considered as a potential new biocatalyst for industrial applications.Fil: Rojas, Natalia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentina. Universidad Nacional de Quilmes; ArgentinaFil: Voget, Claudio Enrique. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Hours, Roque Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; ArgentinaFil: Cavalitto, Sebastian Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Centro de Investigación y Desarrollo en Fermentaciones Industriales. Universidad Nacional de La Plata. Facultad de Ciencias Exactas. Centro de Investigación y Desarrollo en Fermentaciones Industriales; Argentin

Purification and characterization of a keratinolytic serine protease from <i>Purpureocillium lilacinum</i> LPS # 876

A keratinolytic serine protease secreted by Purpureocillium lilacinum (formerly Paecilomyces lilacinus) upon culture in a basal medium containing 1% (w/v) hair waste as carbon and nitrogen source was purified and characterized. After purification the keratinase was resolved by SDS-PAGE as a homogeneus protein band of molecular mass 37.0 kDa. The extracellular keratinase of P. lilacinum was characterized by its appreciable stability over a broad pH range (from 4.0 to 9.0), and up to 65 °C, along with its strong inhibition by phenylmethylsulphonyl fluoride among the protease inhibitors tested (98.2% of inhibition), thus suggesting its nature as a serine protease. The enzyme was active and stable in the presence of organic solvents such as dimethylsulfoxide, methanol, and isopropanol; certain surfactants such as Triton X-100, sodium dodecylsulfate, and Tween 85; and bleaching agents such as hydrogen peroxide. These biochemical characteristics suggest the potential use of this enzyme in numerous industrial applications.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Role of PPase-SE in Geotrichum klebahnii, a yeast-like fungus able to solubilize pectin

Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Role of PPase-SE in Geotrichum klebahnii , a yeast-like fungus able to solubilize pectin

Protopectinases (PPases) constitute a heterogeneous group of extracellular enzymes able to release soluble pectin from insoluble protopectin in plant tissues. Geotrichum klebahnii (ATCC 42397) produces PPase-SE with endopolygalacturonase activity. PPase-SE has been used for pectin extraction and maceration of plant tissues. Here, the capacity of G. klebahnii to use different pectins as carbon and energy sources (CES) was studied, in addition to PPase-SE capacity to release pectin from lemon peel. The strain was unable to use pectin from different origins as CES. When G. klebahnii was cultivated with mixtures of different amounts of glucose and citrus pectin as CES, the biomass obtained was proportional to the initial concentration of glucose, which was completely consumed. In addition, it produced PPase-SE in a glucose-containing medium. A culture was used for the extraction of pectin from lemon peels. Pectin was enzymatically extracted simultaneously with tissue maceration, yielding 3.7 g of (dry) pectin per 100 g of (wet) lemon peel. Extracted pectin was not metabolized by the strain. It was concluded that G. klebahnii uses PPase-SE to macerate, invade and colonize plant tissues, thus releasing soluble sugars to be used as CES without metabolizing solubilized pectin

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Inulinases have been extracted and characterized from inulin-storing tissues,however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.Fil: Ghiringhelli, Pablo Daniel. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Laboratorio de Ingeniería Genética y Biología Molecular y Celular; ArgentinaFil: Hours, Roque Alberto. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Chesini, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Neila, Lorena Paola. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Fratebianchi de la Parra, Dante. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Rojas, Natalia Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: Contreras Esquivel, Juan Carlos. Universidad Autónoma de Coahuila, Facultad de Química; MéxicoFil: Cavalitto, Sebastian Fernando. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); Argentin

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Background: Inulinases have been extracted and characterized from inulin-storing tissues; however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Aspergillus kawachii produces an inulinase in cultures with yacon (Smallanthus sonchifolius) as substrate

Background: Inulinases have been extracted and characterized from inulin-storing tissues; however, production of microbial inulinases have recently draw much attention as they offer several industrial advantages. Many microorganisms, including filamentous fungi, yeast and bacteria have been claimed as inulinase producers. These hydrolases are usually inducible and their exo-acting forms may hydrolyze fructose polymers (inulin) and oligosaccharides such as sucrose and raffinose. Fungal inulinase extracts are often produced as stable mixture of highly active fructanhydrolases. From a practical prospective, the best known inulinases to date are those produced by species of Penicillium, Aspergillus and Kluyveromyces. Results: The production of extracellular inulinase by A. kawachii in liquid cultures, using either inulin or yacon derived materials as CES as well as inulinase inducers, is reported. In addition, a partial characterization of the enzyme activity is included. Conclusions: Yacon derived products, particularly yacon juice, added to the culture medium proved to be a good CES for fungal growth as well as an inducer of enzyme synthesis. Partial characterization of the enzyme revealed that it is quite stable in a wide range of pH and temperature. In addition, characterization of the reaction products revealed that this enzyme corresponds to an exo-type. These facts are promising considering its potential application in inulin hydrolysis for the production of high fructose syrups.Centro de Investigación y Desarrollo en Fermentaciones Industriale

Utilización de yacón para la producción de inulinasas de <i>Aspergillus kawachii</i> en cultivos líquidos sumergidos

Las inulinasas microbianas o β-(2→1) fructanohidrolasas son enzimas que hidrolizan enlaces β-(2→1) fructano en la inulina y se utilizan para la producción de jarabe de fructosa, oligofructosacáridos y etanol o acetona-butanol a partir de residuos de especies vegetales. Estas enzimas pueden clasificarse en exo y endoinulinasas. Las exoinulinasas (β-D-fructan fructanohidrolasa) hidrolizan los enlaces β(2→1) de la mólecula de inulina y separan sucesivamente unidades de frutosa mientras que las endoinulinasas (2,1-β-D fructan fructanohidrolasa) hidrolizan los enlaces internos β(2→1) de inulina y dan como producto final una mezcla de oligofructosacáridos. El Yacón (Smallanthus sonchifolius) es una planta típica de la zona andina (particularmente Jujuy) cuya raíz tiene un alto contenido de inulina y FOS. Actualmente su cultivo se realiza en forma artesanal. El uso del mismo como sustrato para la producción de inulinasas y la posible producción, por medio de dichas enzimas, de jarabes de alta fructosa o soluciones de FOS podría revalorizar un cultivo tradicional del NOA. El objetivo de este trabajo fue estudiar el aprovechamiento de yacón para la producción de inulinasa en cultivos líquidos sumergidos. Se realizaron cultivos de A. kawachii IFO 4308 en medio Czapek líquido con glucosa, fructosa, sacarosa, inulina o jugo de yacón como fuente de carbono y energía. El jugo de yacón se obtuvo por prensado de los frutos frescos, es esterilizó por filtración y se guardó a 4ºC hasta su utilización. Los cultivos se realizaron en erlenmeyers de 1000 ml conteniendo 200 ml de medio, se inocularon con 10⁶ esporos por ml y se incubaron a 200 RPM a 30ºC durante 150h. En el caso de los cultivos con yacón como FCE, se estudió también el efecto del pH inicial. La actividad inulinasa se determinó midiendo la liberación de grupos reductores a partir de una solución 0.05% de inulina a pH 5 en buffer BCP. Con yacón como FCE se obtuvo la mayor actividad, por lo que fue seleccionado para estudios posteriores de producción (en términos de condiciones de cultivo tales como el pH) y caracterización de la enzima con actividad inulinasa. El valor óptimo de pH inicial fue 3 alcanzándose un valor máximo de producción de enzima (50 mU/ml) a las 40 h cuando el pH, debido al consumo de la fuente de nitrógeno (NaNO₃), alcanza un valor de 6. De estos resultados puede concluirse que el yacón resulta una buena alternativa como FCE para la producción de inulinasa en medio líquido.Centro de Investigación y Desarrollo en Fermentaciones Industriale

IDE ‑OTALEX C: A Primeira Infraestrutura de Dados Espaciais transfronteiriça entre Portugal e Espanha

Em 2007 criou ‑se a primeira Infraestrutura de Dados Espaciais transfronteiriça entre Portugal e Espanha (IDE ‑OTALEX – www.ideotalex.eu), que constituiu o Observatório Territorial e Ambiental Alentejo e Extremadura, ao qual se incorporou, em 2011, a região Centro de Portugal, que no total abrange uma superfície de 92.500 km2. Assim, surgiu o Observatório Territorial Alentejo ‑Extremadura ‑Centro (OTALEX C), possibilitando a integração da informação produzida pelas diversas instituições que desenvolvem as suas competências de planeamento e gestão territorial, nestas três regiões. Tendo como objetivo a monitorização e análise de alterações decorrentes de fenómenos naturais e da atividade humana sobre o território, bem como a disponibilização de dados e indicadores aos agentes que atuam neste território, foi desenvolvido um sistema de indicadores comuns, distribuídos por cinco vetores (territorial, ambiental, social, económico e de sustentabilidade). Os dados sofreram trabalhos de homogeneização e estandardização antes de serem integrados tendo em vista facilitar a visualização de mapas, consulta de topónimos e de catálogo, no âmbito da Diretiva INSPIRE. A IDE ‑OTALEX C é o resultado do esforço, do compromisso e da colaboração entre instituições da fronteira, com implicação aos três níveis administrativos: Nacional, Regional e Local. Concede uma visão sobre a situação real do território, ao mesmo tempo que faculta instrumentos adequados para as políticas de ação, que contribuem para apoiar o planeamento e ordenamento do território, a fim de alcançar um desenvolvimento sustentável.info:eu-repo/semantics/acceptedVersio