Purificación y caracterización de poligalacturonasas de Aspergillus kawachii

El desarrollo de nuevas aplicaciones de las pectinasas depende de la búsqueda de enzimas que catalizen reacciones novedosas o que presenten propiedades diferentes a las ya estudiadas. Considerando la capacidad de A. kawachii de producir enzimas activas en condiciones de alta acidez y al escaso conocimiento de su sistema pectolítico, se inició un proyecto de investigación dirigido a estudiar las pectinasas de este microorganismo. El presente trabajo de Tesis se enmarcó en los objetivos de dicho proyecto y se relacionó con el screening de actividades enzimáticas, la purificación y caracterización de una PGasa activa sobre sustratos solubles e insolubles a valores bajos de pH (2.0-3.0) y su aplicación a la extracción enzimática de pectina y la maceración de tejidos. El trabajo se extendió a la purificación y caracterización de poligalacturonasas no-ácidas y al estudio del fenómeno de adsorción selectiva de estas PGasas a filtros de fibra de vidrio. Dicho fenómeno fué observado durante los estudios preliminares de screening y constituyó un aporte original no previamente contemplado en los objetivos iniciales del proyecto. A continuación se detalla el plan desarrollado en el presente trabajo. - Screening de enzimas pectolíticas (Capítulo 2) Cultivos de A. kawachii en medios líquidos con diferentes fuentes de carbono para el screening de enzimas. Desarrollo de un test en placa y zimograma para detectar actividad frente a RG. Caracterización preliminar de la actividad solubilizadora de pectina. - Purificación y propiedades de una poligalacturonasa ácida (Capítulo 3) Purificación de una PGasa ácida (PGI) parcialmente constitutiva producida en un medio con glucosa. Determinación de sus propiedades fisico-químicas y cinéticas, modo de acción (exo/endo), estabilidad térmica y especificidad de sustrato. - Empleo de poligalacturonasas en procesos de extracción de pectina y maceración de tejidos vegetales (Capítulo 4) Ensayos de maceración de frutas y hortalizas y extracción de pectina de pomaza de limón con la PGI de A. kawachii. Comparación de la pectina extraída enzimáticamente a pH ácido con pectinas extraídas por métodos químicos y con PGasa de A. niger. Adsorción de PGasas no-ácidas de A. kawachii a filtros de fibra de vidrio. - Purificación y propiedades de las PGasas (Capítulo 5) Estudio del fenómeno de adsorción de pectinasas a filtros de fibra de vidrio. Purificación y caracterización de las PGasas involucradas en el proceso de adsorción.Facultad de Ciencias Exacta

Evaluation of Culture Conditions for Tannase Production by Aspergillus niger GH1

Ispitana je ekstracelularna i intracelularna proizvodnja tanaze submerznim uzgojem plijesni Aspergillus niger GH1 u tekućoj te površinskim uzgojem na čvrstoj podlozi pri različitim temperaturama (30, 40 i 50 °C). Istražen je utjecaj koncentracije početnog supstrata (taninske kiseline), vremena inkubacije i temperature na proizvodnju tanaze pri uzgoju na čvrstoj podlozi. Soj A. niger GH1 proizveo je najviše tanaze (2291 U/L) tijekom prvih 20 sati uzgoja pri koncentraciji taninske kiseline od 50 g/L. U tim uvjetima proizvodnje enzim je u potpunosti bio ekstracelularan. Opadanje aktivnosti tanaze nakon 20 sati ugoja povezano je s pratećim porastom aktivnosti proteaze.Extra- and intracellular tannase production by Aspergillus niger GH1 has been evaluated using submerged (SmF) and solid-state fermentation (SSF) at different temperatures (30, 40 and 50 °C). Effects of initial substrate (tannic acid) concentration, incubation time and temperature on tannase production in SSF have been studied. A. niger GH1 produced the highest tannase level (2291 U/L) in SSF at 30 °C during the first 20 h of culture at tannic acid concentration of 50 g/L, and under these conditions enzyme production was entirely extracellular. The decline in tannase activity after 20 h of incubation was associated with a concomitant increase in protease activity

Isolation and Evaluation of Tannin-degrading Fungal Strains from the Mexican Desert

Eleven fungal strains (4 Penicillium commune, 2 Aspergillus niger, 2 Aspergillus rugulosa, Aspergillus terricola, Aspergillus ornatus and Aspergillus fumigatus) were isolated, characterized morphologically and by their capacity to degrade tannins. Aspergillus niger Aa-20 was used as control strain. Several concentrations of hydrolysable tannin (tannic acid) were used as sole carbon source. All strains were able to degrade hydrolysable tannins. Aspergillus niger GH1 and PSH showed the highest tannin-degrading capacity (67 and 70%, respectively). Also, the fungal capacity to degrade condensed tannin (catechin) was tested. Aspergillus niger PSH and Penicillium commune EH2 degraded 79.33% and 76.35% of catechin. The results demonstrated the capacity of fungi to use hydrolysable and condensed tannins as carbon source

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

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

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