Inulinasa de <i>Aspergillus kawachii</i> IFO 4308: caracterización, clonado, sobreexpresión, y aplicación en procesos biotecnológicos

El presente trabajo de tesis doctoral describe la caracterización, clonado, sobreexpresión, y aplicación en procesos biotecnológicos de una inulinasa de Aspergillus kawachii IFO 4308. Las inulinasas son enzimas que actúan sobre los enlaces β-(2→1) fructano en la inulina presentes en muchos vegetales. Es por ello, que el aprovechamiento de la actividad catalítica de estas enzimas es de gran interés en numerosas aplicaciones biotecnológicas como la producción de fructosa, de fructooligosacáridos, bioetanol, 2,3-butanodiol, ácido láctico, jarabe de sorbitol, ácido glucónico, manitol, entre otros. Los microorganismos son las mejores fuentes para la producción de inulinasas debido a su fácil cultivo y alta producción de enzima, en comparación con los vegetales. El género Aspergillus ha sido reportado como uno de los mayores productores de inulinasa. En general, estos microorganismos que producen enzimas de interés industrial lo hacen en niveles muy bajos, por lo tanto, se hace necesario incrementar estos rendimientos para lograr una mayor rentabilidad de los procesos. Una forma de mejorar el rendimiento es mediante la optimización del medio de cultivo y de las condiciones de operación, pero esto está limitado por la capacidad de síntesis máxima del producto deseado que tiene el organismo. Otra posibilidad es la producción de la enzima en sistemas recombinantes para lograr la obtención de altas cantidades requeridas en la industria. El sistema de expresión de Pichia pastoris ha mostrado gran eficiencia en la producción de enzimas y fue el utilizado en este trabajo de tesis. La presencia de inulina en los tubérculos de yacón despertó el interés por utilizar esta raíz andina, cultivada en el norte argentino, como sustrato para la producción de la enzima, como alternativa a las materias primas comerciales actuales y en el horizonte de revalorar la producción regional de este producto. Se comenzó con el estudio de la producción de inulinasa utilizando la cepa Aspergillus kawachii IFO 4308. Para aumentar la producción de la enzima se estudió el efecto de diferentes fuentes de carbono y energía individualmente o en combinación con yacón, fuentes de nitrógeno, así como los parámetros del proceso: concentración de inóculo, temperatura y pH inicial. Se utilizó una estrategia de optimización del medio de cultivo de una variable a la vez, así se logró duplicar la actividad enzimática, que alcanzó un valor de 92,3±0,5 mU/ml a las 72 h de cultivo (productividad = 1,28 U/l/h). En escala de biorreactor la actividad inulinasa fue de 124,1 ±2,1 mU/ml a las 48 h de cultivo (productividad= 2,58 U/l/h), en las mismas condiciones, lo que significó un incremento de 1,3 veces con respecto al cultivo en matraz Erlenmeyer. La inulinasa producida por Aspergillus kawachii IFO 4308 presentó pH óptimo de 3 y resultó ser estable a una temperatura de 65 °C por 180 minutos, donde retuvo el 20 % de la actividad residual. Estas características plantearon la posibilidad de estudiar su aplicación en procesos industriales, sin embargo, la baja productividad del proceso completo llevó a considerar una estrategia de clonado y la sobreexpresión para hacer este proceso más adecuado para sus potenciales demandas y aplicaciones industriales. Se llevaron adelante tres estrategias de clonado, una a partir de ARN y dos a partir de ADN, una utilizando el gen inulinasa con intrón en pPIC9 y la otra utilizando el gen inulinasa sin intrón, eliminado in vitro, en pPICZαA. Con esta última estrategia se logró expresar la inulinasa genéticamente modificada que se produjo con éxito en fermentaciones en biorreactor, alcanzando 622,4 U/ml y una productividad de 25933,3 U/l/h después de 69 h un cultivo. Resultó funcional con niveles de producción de actividad enzimática 5000 veces más a los reportados por el organismo silvestre. La enzima recombinante resultó una exoinulinasa con actividad fructosiltransferasa y presentó las mismas propiedades bioquímicas que la enzima nativa. Con la finalidad de mejorar tanto las propiedades de la enzima, como la productividad del proceso, se estudió la inmovilización covalente de la inulinasa recombinante en esferas de quitosan optimizando la concentración de un agente entrecruzante, glutaraldehído, y el tiempo de activación de las esferas mediante un diseño factorial. Se consiguió aumentar la estabilidad operacional de la enzima que retuvo 40 % actividad residual a 65 °C durante 180 minutos, lo que posibilita una ventaja adicional en su campo de acción. El enlace formado entre la inulinasa recombinante y el quitosán resultó ser lo suficientemente estable para evitar desprendimientos de la enzima y con ello mantener la eficiencia catalítica del sistema durante 12 ciclos de uso. Finalmente, se estudiaron las condiciones de aplicación, la producción de FOS partir de sacarosa y la hidrólisis de fructano de agave a partir de subproductos de la industria. Se obtuvieron altos rendimientos de hidrólisis en jugos de agave. La combinación de estrategias adecuadas de ingeniería genética, los estudios de producción en escala de biorreactor, los procesos de purificación y los ensayos de inmovilización junto con las características bioquímicas que presentó la enzima, permitieron generar cantidades suficientes de una enzima de aplicación industrial. Estos estudios son la base para un futuro desarrollo de procesos tecnológicos respecto a la obtención de fructooligosacáridos y jarabe de fructosa utilizando una tecnología amigable con el ambiente. Posteriores estudios de optimización, producción a mayor escala y estimación de los costos del proceso completo serán necesarios para la implementación de estos procesos en la industria.Facultad de Ciencias Exacta

Study of the production of alkaline keratinases in submerged cultures as an alternative for solid waste treatment generated in leather technology

Six nonpathogenic fungal strains isolated from alkaline soils of Buenos Aires Province, Argentina (Acremonium murorum, Aspergillus sidowii, Cladosporium cladosporoides, Neurospora tetrasperma, Purpureocillium lilacinum (formerly Paecilomyces lilacinus), and Westerdikella dispersa) were tested for their ability to produce keratinolytic enzymes. Strains were grown on feather meal agar as well as in solid-state and submerged cultures, using a basal mineral medium and “hair waste” as sole sources of carbon and nitrogen. All the tested fungi grew on feather meal agar, but only three of them were capable of hydrolyzing keratin, producing clear zones. Among these strains, P. lilacinum produced the highest proteolytic and keratinolytic activities, both in solid-state and submerged fermentations. The medium composition and culture conditions for the keratinases production by P. lilacinum were optimized. Addition of glucose (5 g/l) and yeast extract (2.23 g/l) to the basal hair medium increased keratinases production. The optimum temperature and initial pH for the enzyme production were 28o C and 6.0, respectively. A beneficial effect was observed when the original concentration of four metal ions, present in the basal mineral medium, was reduced up to 1:10. The maximum yield of the enzyme was 15.96 Uc/ml in the optimal hair medium; this value was about 6.5-fold higher than the yield in the basal hair medium. These results suggest that keratinases from P. lilacinum can be useful for biotechnological purposes such as biodegradation (or bioconversion) of hair waste, leading to a reduction of the environmental pollution caused by leather technology with the concomitant production of proteolytic enzymes and protein hydrolyzatesFil: Chesini, Mariana. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico la Plata. Centro de Investigación y Desarrollo En Fermentaciones Industriales (i); ArgentinaFil: 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: 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

Optimiranje proizvodnje poligalakturonaze iz Aspergillus kawachii, klonirane u Saccharomyces cerevisiae, u šaržnom i prihranjivanom šaržnom uzgoju

Polygalacturonases (PG; EC 3.2.1.15) catalyze the hydrolysis of pectin and/or pectic acid and are useful for industrial applications such as juice clarification and pectin extraction. Growth and heterologous expression of recombinant Saccharomyces cerevisiae which expresses an acidic PG from Aspergillus kawachii has been studied in batch and fed-batch cultures. Kinetics and stoichiometric parameters of the recombinant yeast were determined in batch cultures in a synthetic medium. In these cultures, the total biomass concentration, protein concentration, and enzyme activity achieved were 2.2 g/L, 10 mg/L, and 3 U/mL, respectively, to give a productivity of 0.06 U/(mL·h). In fed-batch cultures, various strategies for galactose feeding were used: (i) after a glucose growth phase, the addition of a single pulse of galactose which gave a productivity of 0.19 U/(mL·h); (ii) after a glucose growth phase, a double pulse of galactose at the same final concentration was added, resulting in a productivity of 0.21 U/(mL·h); (iii) a simultaneous feeding of glucose and galactose, yielding a productivity of 1.32 U/(mL·h). Based on these results, the simultaneous feeding of glucose and galactose was by far the most suitable strategy for the production of this enzyme. Moreover, some biochemical characteristics of the recombinant enzyme such as a molecular mass of ~60 kDa, an isoelectric point of 3.7 and its ability to hydrolyze polygalacturonic acid at pH=2.5 were determined.Poligalakturonaze (EC 3.2.1.15) kataliziraju hidrolizu pektina i/ili pektinske kiseline i mogu se primijeniti u prehrambenoj industriji, npr. za bistrenje soka ili ekstrakciju pektina. U radu je ispitan rast rekombinantnog kvasca Saccharomyces cerevisiae, te heterologna ekspresija kisele poligalakturonaze iz Aspergillus kawachii u šaržnom i prihranjivanom šaržnom uzgoju. U sintetičkom su mediju šaržnim uzgojem određeni kinetički i stehiometrijski parametri rekombinantnog kvasca. Pritom su dobivene ove vrijednosti: koncentracija ukupne biomase od 2,2 g/L; koncentracija proteina od 10 mg/L; enzimska aktivnost od 3 U/mL i produktivnost od 0,006 U/(mL·h). U prihranjivanom su šaržnom uzgoju primijenjene razne strategije prihranjivanja galaktozom: (i) nakon što kvasac utroši glukozu za rast, jednokratnim dodatkom galaktoze postiže se produktivnost od 0,19 U/(mL·h); (ii) nakon utroška glukoze, dvaput se doda galaktoza u jednakim koncentracijama, pri čemu je produktivnost 0,21 U/(mL·h) i (iii) simultanim prihranjivanjem glukozom i galaktozom dobivena je produktivnost od 1,32 U/(mL·h). Zaključeno je da se najbolji rezultati postižu simultanim prihranjivanjem glukozom i galaktozom. Određene su i biokemijske značajke rekombinantnog enzima: molekularna masa od približno 60 kDa, izoelektrična točka od 3,7 i sposobnost enzima da hidrolizira poligalakturonsku kiselinu pri pH=2,5

Optimization of the Production of a Polygalacturonase from Aspergillus kawachii cloned in Saccharomyces cerevisiae in batch and fed-batch cultures

Polygalacturonases (PG; EC 3.2.1.15) catalyze the hydrolysis of pectin and/or pectic acid and are useful for industrial applications such as juice clarification and pectin extraction. Growth and heterologous expression of recombinant S. cerevisiae which expresses an acidic PG from Aspergillus kawachii was studied in batch and fed batch cultures. Kinetics and stoichiometric parameters of the recombinant yeast were determined in batch cultures in a synthetic medium. In these cultures, the total biomass concentration, protein concentration, and enzyme activity achieved were 2.2 g/l, 10 mg/l, and 3 U/ml, respectively, to give a productivity of 0.06 U/ml.h. In fed batch cultures, various strategies for galactose feeding were used: a) after a glucose growth phase, the addition of a single pulse of galactose which gave a productivity of 0.19 U/ml.h.; b) after a glucose growth phase, a double pulse of galactose at the same final concentration, resulting in a productivity of 0.21 U/ml.h.; c) a simultaneous feeding of glucose and galactose, yielding a productivity of 1.32 U/ml.h. Based on these results, the simultaneous feeding of glucose and galactose was by far the most suitable strategy for the production of this enzyme. Moreover, some biochemical characteristics of the recombinant enzyme such as a MW of ~60 kDa, an isoelectric point of 3.7 and its ability to hydrolyze polygalacturonic acid at pH 2.5 were determined.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; ArgentinaFil: Ortiz, Gastón Ezequiel. 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: Chesini, Mariana. 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: Baruque, Diego Jorge. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; 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

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

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

Optimization of the Production of Polygalacturonase from Aspergillus kawachii Cloned in Saccharomyces cerevisiae in Batch and Fed-Batch Cultures

Polygalacturonases (PG; EC 3.2.1.15) catalyze the hydrolysis of pectin and/or pectic acid and are useful for industrial applications such as juice clarification and pectin extraction. Growth and heterologous expression of recombinant Saccharomyces cerevisiae which expresses an acidic PG from Aspergillus kawachii has been studied in batch and fed-batch cultures. Kinetics and stoichiometric parameters of the recombinant yeast were determined in batch cultures in a synthetic medium. In these cultures, the total biomass concentration, protein concentration, and enzyme activity achieved were 2.2 g/L, 10 mg/L, and 3 U/mL, respectively, to give a productivity of 0.06 U/(mL·h). In fed-batch cultures, various strategies for galactose feeding were used: (i) after a glucose growth phase, the addition of a single pulse of galactose which gave a productivity of 0.19 U/(mL·h); (ii) after a glucose growth phase, a double pulse of galactose at the same final concentration was added, resulting in a productivity of 0.21 U/(mL·h); (iii) a simultaneous feeding of glucose and galactose, yielding a productivity of 1.32 U/(mL·h). Based on these results, the simultaneous feeding of glucose and galactose was by far the most suitable strategy for the production of this enzyme. Moreover, some biochemical characteristics of the recombinant enzyme such as a molecular mass of ~60 kDa, an isoelectric point of 3.7 and its ability to hydrolyze polygalacturonic acid at pH=2.5 were determined