RESUMEN
AIMS: The aim of this work was to characterize and apply a polygalacturonase of Penicillium janthinellum new strain VI2R3M. METHODS AND RESULTS: The polygalacturonase obtained from P. janthinellum VI2R3M was incubated in cultures of passion fruit peel and was partially purified by ion-exchange chromatography and gel filtration. The enzyme showed a relative molecular mass of 102·0 kDa, maximum activity at pH 5·0, temperature of 50°C, 100% stablity at 50°C and 80% stablity at pH 3·0-5·0. The apparent Km , Vmax and Kcat values for hydrolyzing polygalacturonic acid were 2·56 mg ml-1 , 163·1 U mg-1 and 277 s-1 respectively. The polygalacturonase presented exo activity and was activated by Mg2+ . The juices treated with polygalacturonase presented increases in transmittance with reduction in colour. CONCLUSIONS: The results suggest that the new lineage P. janthinellum VI2R3M presents a high yield of an exo-polygalacturonase induced by agro-industrial residues, with excellent activity and stability in acidic pH and at 50°C. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of agro-industrial residue to obtain the polygalacturonase can contribute to a decrease enzyme production cost. The results of the activity, stability to acidic pH and excellent performance in the clarification of juices show that the enzyme is promising for industrial application.
Asunto(s)
Jugos de Frutas y Vegetales , Penicillium/enzimología , Poligalacturonasa/química , Poligalacturonasa/metabolismo , Biotecnología , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Hidrólisis , Peso Molecular , Pectinas/metabolismo , Penicillium/metabolismo , Poligalacturonasa/aislamiento & purificación , TemperaturaRESUMEN
Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources. Bacillus licheniformis has been used as an important source for bioprospection of several industrial biomolecules, such as surfactants and enzymes, including pectate lyases. Here we cloned, expressed, purified, and biochemically and structurally characterized an exo-polygalacturonase from B. licheniformis (BlExoPG). Its low-resolution molecular envelope was derived from experimental small-angle scattering data (SAXS). Our experimental data revealed that BlExoPG is a monomeric enzyme with optimum pH at 6.5 and optimal temperature of approximately 60°C, at which it has considerable stability over the broad pH range from 5 to 10. After incubation of the enzyme for 30min at pH ranging from 5 to 10, no significant loss of the original enzyme activity was observed. Furthermore, the enzyme maintained residual activity of greater than 80% at 50°C after 15h of incubation. BlExoPG is more active against polygalacturonic acid as compared to methylated pectin, liberating mono galacturonic acid as a unique product. Its enzymatic parameters are Vmax=4.18µM.s-1,Km=3.25mgmL-1 and kcat=2.58s-1.
Asunto(s)
Bacillus licheniformis/enzimología , Poligalacturonasa/química , Dispersión del Ángulo Pequeño , Concentración de Iones de Hidrógeno , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Temperatura , Difracción de Rayos XRESUMEN
An endo-polygalacturonase secreted by Aspergillus sojae was characterized after being purified to homogeneity from submerged cultures with orange peel as the sole carbon source by gel filtration and ion-exchange chromatographies. According to SDS-PAGE and analytical isoelectric focusing analyses, the enzyme presents a molecular weight of 47 kDa and pI value of 4.2. This enzyme exhibits considerable stability under highly acidic to neutral conditions (pH 1.5-6.5) and presents a half-life of 2 h at 50°C. Besides its activity towards pectin and polygalacturonic acid, the enzyme displays pectin-releasing activity, acting best in a pH range of 3.3-5.0. Thin-layer chromatographic analysis revealed that tri-galacturonate is the main enzymatic end product of polygalacturonic acid hydrolysis, indicating that it is an endo-polygalacturonase. The enzyme exhibits Michaelis-Menten kinetics, with KM and VMAX values of 0.134 mg/mL and 9.6 µmol/mg/min, respectively, and remained stable and active in the presence of SO2, ethanol, and various cations assayed except Hg2+.
Asunto(s)
Aspergillus/enzimología , Poligalacturonasa/química , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Carbono/metabolismo , Cromatografía en Gel/métodos , Cromatografía por Intercambio Iónico/métodos , Cromatografía en Capa Delgada/métodos , Citrus sinensis/química , Electroforesis en Gel de Poliacrilamida/métodos , Pruebas de Enzimas/métodos , Estabilidad de Enzimas/efectos de los fármacos , Etanol/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Ácidos Hexurónicos/metabolismo , Concentración de Iones de Hidrógeno , Focalización Isoeléctrica/métodos , Cinética , Metales/metabolismo , Peso Molecular , Pectinas/metabolismo , Dióxido de Azufre/metabolismo , Temperatura , Factores de TiempoRESUMEN
This work reports the production of an exo-polygalacturonase (exo-PG) by Rhizomucor pusillus A13.36 in submerged cultivation (SmC) in a shaker at 45°C for 96 h. A single pectinase was found and purified in order to analyze its thermal stability, by salt precipitation and hydrophobic interaction chromatography. The pectinase has an estimated Mw of approximately 43.5-47 kDa and optimum pH of 4.0 but is stable in pH ranging from 3.5 to 9.5 and has an optimum temperature of 61°C. It presents thermal stability between 30 and 60°C, has 70% activation in the presence of Ca2+, and was tested using citrus pectin with a degree of methyl esterification (DE) of 26%. Ea(d) for irreversible denaturation was 125.5 kJ/mol with positive variations of entropy and enthalpy for that and ΔG(d) values were around 50 kJ/mol. The hydrolysis of polygalacturonate was analyzed by capillary electrophoresis which displayed a pattern of sequential hydrolysis (exo). The partial identification of the primary sequence was done by MS MALDI-TOF and a comparison with data banks showed the highest identity of the sequenced fragments of exo-PG from R. pusillus with an exo-pectinase from Aspergillus fumigatus. Pectin hydrolysis showed a sigmoidal curve for the Michaelis-Menten plot.
Asunto(s)
Proteínas Fúngicas , Poligalacturonasa , Rhizomucor , Estabilidad de Enzimas , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Proteínas Fúngicas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Poligalacturonasa/química , Poligalacturonasa/genética , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Rhizomucor/enzimología , Rhizomucor/genética , Rhizomucor/crecimiento & desarrolloRESUMEN
Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes --#91;pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase--#93; by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3 U/ml and polymethylgalacturonase between 0.15 and 1.3 U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36 U/l and 63 U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.
Macrophomina phaseolina es un fitopatógeno polífago, causante de podredumbre carbonosa. Los daños que genera en cultivos de soja y maíz bajo siembra directa en Argentina, en períodos secos y calurosos, se incrementaron por su habilidad para sobrevivir como esclerocios en suelos y restos de cosecha. El propósito del trabajo fue estudiar la producción in vitro de enzimas degradadoras de pared celular vegetal (pectinasas --#91;poligalacturonasa y polimetilgalacturonasa--#93;; celulasas --#91;endoglucanasa--#93;; hemicelulasas --#91;endoxilanasa--#93; y la enzima ligninolítica lacasa) de varios aislamientos argentinos de M. phaseolina y evaluar la patogenicidad de esos aislamientos, como paso preliminar para establecer el papel de estas enzimas en la interacción M. phaseolina-maíz. Se estudió la cinética de crecimiento del hongo y la de la producción de dichas enzimas en medios de cultivo líquidos sintéticos con ácido glutámico como fuente de nitrógeno y con pectina, carboximetilcelulosa (CMC) o xilano como fuentes de carbono. Las pectinasas fueron las primeras enzimas detectadas y los máximos títulos registrados (1,4 UE/ml --#91;poligalacturonasa--#93; y 1,2 UE/ml --#91;polimetilgalacturonasa--#93;, respectivamente) superaron a los de celulasas y xilanasas, que aparecieron más tardíamente y en menor magnitud. Esta secuencia promovería la maceración inicial del tejido, seguida luego por la degradación de la pared celular vegetal. Se detectó actividad lacasa en todos los aislamientos (36 a 63 U/l). La agresividad de todos los aislamientos resultó alta en los 3 hospedantes evaluados: semillas de maíz, de girasol y de melón. En este trabajo se investiga por primera vez el potencial de distintos aislamientos de M. phaseolina para producir enzimas degradadoras de pared celular vegetal en cultivo líquido.
Asunto(s)
Técnicas In Vitro/métodos , Pared Celular/enzimología , Zea mays/enzimología , Zea mays/parasitología , Poligalacturonasa/aislamiento & purificación , Celulasa/aislamiento & purificación , Endo-1,4-beta Xilanasas/aislamiento & purificaciónRESUMEN
Macrophomina phaseolina is a polyphagous phytopathogen, causing stalk rot on many commercially important species. Damages caused by this pathogen in soybean and maize crops in Argentina during drought and hot weather have increased due its ability to survive as sclerotia in soil and crop debris under non-till practices. In this work, we explored the in vitro production of plant cell wall-degrading enzymes [pectinases (polygalacturonase and polymethylgalacturonase); cellulases (endoglucanase); hemicellulases (endoxylanase) and the ligninolytic enzyme laccase] by several Argentinean isolates of M. phaseolina, and assessed the pathogenicity of these isolates as a preliminary step to establish the role of these enzymes in M. phaseolina-maize interaction. The isolates were grown in liquid synthetic medium supplemented with glucose, pectin, carboxymethylcellulose or xylan as carbon sources and/or enzyme inducers and glutamic acid as nitrogen source. Pectinases were the first cell wall-degrading enzymes detected and the activities obtained (polygalacturonase activity was between 0.4 and 1.3U/ml and polymethylgalacturonase between 0.15 and 1.3U/ml) were higher than those of cellulases and xylanases, which appeared later and in a lesser magnitude. This sequence would promote initial tissue maceration followed by cell wall degradation. Laccase was detected in all the isolates evaluated (activity was between 36U/l and 63U/l). The aggressiveness of the isolates was tested in maize, sunflower and watermelon seeds, being high on all the plants assayed. This study reports for the first time the potential of different isolates of M. phaseolina to produce plant cell wall-degrading enzymes in submerged fermentation.
Asunto(s)
Ascomicetos/enzimología , Proteínas Fúngicas/metabolismo , Argentina , Ascomicetos/crecimiento & desarrollo , Ascomicetos/aislamiento & purificación , Ascomicetos/patogenicidad , Carbono/metabolismo , Pared Celular/metabolismo , Celulasa/aislamiento & purificación , Celulasa/metabolismo , Citrullus/microbiología , Medios de Cultivo , Endo-1,4-beta Xilanasas/aislamiento & purificación , Endo-1,4-beta Xilanasas/metabolismo , Fermentación , Proteínas Fúngicas/aislamiento & purificación , Glicósido Hidrolasas/aislamiento & purificación , Glicósido Hidrolasas/metabolismo , Helianthus/microbiología , Microbiología Industrial/métodos , Lacasa/aislamiento & purificación , Lacasa/metabolismo , Nitrógeno/metabolismo , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Semillas/microbiología , Zea mays/microbiologíaRESUMEN
A soil isolate, Penicillium janthinellum sw09 has been found to produce significant amounts of an extracellular pectinase subsequently characterized as exo-polygalacturonase (exo-PG). By optimizing growth conditions, P. janthinellum sw09 produced high amount of exo-PG (16.54 units/mL). The crude enzyme was purified by gel filtration chromatography and two exo-PG activity peaks (designated as PGI and PGII) were revealed. On SDS-PAGE analysis, purified PGII using DEAE-Sepharose FF column, was found to be a single band with a molecular mass of 66.2 kDa. The purified PGII exhibited maximal activity at the temperature of 45 oC and pH 5.0. The stability profiles show that PGII is more stable in the pH range of 4.0-8.0 and below 60 oC. The Km and Vmax for the enzyme was 1.74 mg/mL and 18.08 µmol/ (mLâ¢min), respectively. Due to this enzymatic characterization, this pectinase is an attractive candidate for applications in degradation of pectin.
Asunto(s)
Penicillium/enzimología , Poligalacturonasa/biosíntesis , Electroforesis en Gel de Poliacrilamida , Concentración de Iones de Hidrógeno , Penicillium/crecimiento & desarrollo , Poligalacturonasa/química , Poligalacturonasa/aislamiento & purificación , TemperaturaRESUMEN
Recently, it has been suggested that pectinases could be used to hydrolyze pectin in biorefineries based on pectin-rich agro-industrial wastes. However, for this to be viable, the cost of their production would need to be lowered significantly. In fact, over the last few decades, there have been many attempts to improve pectinase production by existing strains or to screen for new strains from environmental isolates. In these studies, it is necessary to measure pectinase activities. Many researchers use single-time-point assays that involve incubation of pectinolytic extracts with pectic substrates for a fixed time, followed by determination of the liberated reducing sugars. However, different researchers use quite different conditions for this assay. Furthermore, no attention has been given to the reaction profile during the assay. In the current work, we show, for the first time, that a significant deceleration of the rate of liberation of reducing sugars occurs over the first ten minutes of the reaction. As a consequence, the incubation time used in a single-time-point assay has a large effect on the value obtained for the activity. In fact, we demonstrate that, depending on the particular combination of incubation time, pectin concentration and reaction temperature, the same extract could be reported to have activities that differ by an order of magnitude. In addition, we show that the relative activities obtained with polygalacturonic acid do not correlate with those obtained with pectin. We conclude that it is currently impossible to make meaningful comparisons between pectinase activities reported in the literature by workers who have used different assay conditions. Therefore there is an urgent need for the development of a standardized assay for evaluating the saccharification potential of pectinase complexes.
Asunto(s)
Aspergillus/química , Fermentación , Residuos Industriales , Pectinas/química , Celulasas/química , Hidrólisis , Poligalacturonasa/química , Poligalacturonasa/aislamiento & purificación , Tiempo de ReacciónRESUMEN
Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T(660 nm) = 97.0%) of juice was attained after 2 h of incubation at 50 °C in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.
Asunto(s)
Aspergillus/enzimología , Bebidas , Manipulación de Alimentos/métodos , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , alfa-Amilasas/aislamiento & purificación , alfa-Amilasas/metabolismo , Aspergillus/crecimiento & desarrollo , Medios de Cultivo/química , Depuradores de Radicales Libres/análisis , Fenoles/análisis , Temperatura , Factores de TiempoRESUMEN
Since enzymatic degradation is a mechanism or component of the aggressiveness of a pathogen, enzymatic activities from a Fusarium graminearum isolate obtained from infected wheat spikes of Argentina Pampa region were studied in order to understand the disease progression, tending to help disease control. In particular, the significance of the study of polygalacturonase activity is based on that such activity is produced in the early stages of infection on the host, suggesting a crucial role in the establishment of disease. In this sense, polygalacturonase activity produced by this microorganism has been purified 375 times from 2-day-old culture filtrates by gel filtration and ion-exchange chromatography successively. The purified sample showed two protein bands in sodium dodecyl sulfate-polyacrylamide gels, with a molecular mass of 40 and 55 kDa. The protein bands were identified as an endopolygalacturonase and as a serine carboxypeptidase of F. graminearum, respectively, by peptide mass fingerprinting (matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF/TOF) fragment ion analysis). The pattern of substrate degradation analyzed by thin layer chromatography confirmed the mode of action of the enzyme as an endopolygalacturonase. High activity of the polygalacturonase against polygalacturonic acid was observed between 4 and 6 of pH, and between 30 and 50 °C, being 5 and 50 °C the optimum pH and temperature, respectively. The enzyme was fully stable at pH 5 for 120 min and 30 °C and sensible to the presence of some metal ions. This information would contribute to understand the most favorable environmental conditions for establishment of the disease.
Asunto(s)
Fusarium/enzimología , Pectinas/metabolismo , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Proteoma/análisis , Argentina , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas , Fusarium/química , Fusarium/aislamiento & purificación , Concentración de Iones de Hidrógeno , Peso Molecular , Enfermedades de las Plantas/microbiología , Poligalacturonasa/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Temperatura , Triticum/microbiologíaRESUMEN
Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.
Asunto(s)
Aspergillus/enzimología , Bebidas , Manipulación de Alimentos/métodos , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , alfa-Amilasas/aislamiento & purificación , alfa-Amilasas/metabolismo , Aspergillus/crecimiento & desarrollo , Medios de Cultivo/química , Depuradores de Radicales Libres/análisis , Fenoles/análisis , Temperatura , Factores de TiempoRESUMEN
Polygalacturonase and α-amylase play vital role in fruit juice industry. In the present study, polygalacturonase was produced by Aspergillus awamori Nakazawa MTCC 6652 utilizing apple pomace and mosambi orange (Citrus sinensis var mosambi) peels as solid substrate whereas, α-amylase was produced from A. oryzae (IFO-30103) using wheat bran by solid state fermentation (SSF) process. These carbohydrases were decolourized and purified 8.6-fold, 34.8-fold and 3.5-fold, respectively by activated charcoal powder in a single step with 65.1%, 69.8% and 60% recoveries, respectively. Apple juice was clarified by these decolourized and partially purified enzymes. In presence of 1% polygalacturonase from mosambi peels (9.87 U/mL) and 0.4% α-amylase (899 U/mL), maximum clarity (%T660nm = 97.0%) of juice was attained after 2 h of incubation at 50 ºC in presence of 10 mM CaCl2. Total phenolic content of juice was reduced by 19.8% after clarification, yet with slightly higher %DPPH radical scavenging property.(AU)
Asunto(s)
Aspergillus/enzimología , Bebidas , Manipulación de Alimentos/métodos , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , alfa-Amilasas/aislamiento & purificación , alfa-Amilasas/metabolismo , Aspergillus/crecimiento & desarrollo , Medios de Cultivo/química , Depuradores de Radicales Libres/análisis , Fenoles/análisis , Temperatura , Factores de TiempoRESUMEN
A thermotolerant fungus identified as Aspergillus niveus was isolated from decomposing materials and it has produced excellent levels of hydrolytic enzymes that degrade plant cell walls. A. niveus germinated faster at 40 °C, presenting protein levels almost twofold higher than at 25 °C. The crude extract of the A. niveus culture was purified by diethylaminoethyl (DEAE)-cellulose, followed by Biogel P-100 column. Polygalacturonase (PG) is a glycoprotein with 37.7 % carbohydrate, molecular mass of 102.6 kDa, and isoelectric point of 5.4. The optimum temperature and pH were 50 °C and 4.0-6.5, respectively. The enzyme was stable at pH 3.0 to 9.0 for 24 h. The DEAE-cellulose derivative was about sixfold more stable at 60 °C than the free enzyme. Moreover, the monoaminoethyl-N-aminoethyl-agarose derivative was tenfold more stable than the free enzyme. PG was 232 % activated by Mn(2+). The hydrolysis product of sodium polypectate corresponded at monogalacturonic acid, which classifies the enzyme as an exo-PG. The K m, V max, K cat, and K cat/K m values were 6.7 mg/ml, 230 U/mg, 393.3/s, and 58.7 mg/ml/s, respectively. The N-terminal amino acid sequence presented 80 % identity with PglB1, PglA2, and PglA3 putative exo-PG of Aspergillus fumigatus and an exo-PG Neosartorya fischeri.
Asunto(s)
Aspergillus/enzimología , Activadores de Enzimas/metabolismo , Manganeso/metabolismo , Poligalacturonasa/metabolismo , Aspergillus/crecimiento & desarrollo , Aspergillus/aislamiento & purificación , Análisis por Conglomerados , Microbiología Ambiental , Estabilidad de Enzimas , Ácidos Hexurónicos/metabolismo , Concentración de Iones de Hidrógeno , Punto Isoeléctrico , Cinética , Peso Molecular , Filogenia , Poligalacturonasa/química , Poligalacturonasa/aislamiento & purificación , Homología de Secuencia de Aminoácido , TemperaturaRESUMEN
One of the recurrent methodological problems in preparative biochemical work is the concentration of dilute protein solutions, including culture supernatants resulting from biotechnological processes. A procedure was developed to concentrate enzymes by a novel cryoconcentration system. This approach includes a new device that facilitates the sample freezing and the subsequent solute elution from the frozen matrix by centrifugation. The optimal centrifugation conditions for this cryoconcentration system were obtained using whey protein solution as a model. The procedure was applied to concentrate dilute solutions of commercial pectinase, measuring the endopolygalacturonase (EPG) activity of this enzyme in the concentrate by a method based on the on-line torque measurement, and of recombinant fructan:fructan 1-fructosyltransferase (1-FFT) protein of Pichia pastoris from a culture in a bioreactor, as an expression system. The optimal centrifugation speed, time, and temperature were 6150 g, 20 min, and 4 °C, respectively. The concentration factors for the dilute protein solutions were 9.2-, 11.2-, and 17.1-fold for 1-FFT, whey, and commercial pectinase, respectively. Recoveries ranged from 87% to 93%. The procedure allowed concentrating proteins efficiently without affecting their enzymatic activity.
Asunto(s)
Hexosiltransferasas/metabolismo , Poligalacturonasa/metabolismo , Centrifugación , Diálisis , Congelación , Hexosiltransferasas/genética , Hexosiltransferasas/aislamiento & purificación , Pichia/metabolismo , Poligalacturonasa/aislamiento & purificación , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The present study was undertaken with the purpose of selecting yeasts from wine grapes that are able to produce extracellular cold-active pectinases. After two consecutive selections yeast isolates were identified by pheno- and genotyping, and pectinolytic activity was preliminarily characterised at proximate winemaking conditions. Out of 1023 indigenous microorganisms isolated from grape skins of D.O. San Rafael (Mendoza, Argentina) viticulture region, 565 (55%) showed pectinolytic activity on plates and, among them, 96 (17%) were chosen in a primary selection. Ten isolates were finally selected for exhibiting the greatest activity at low temperature (12 °C) and identified as Aureobasidium pullulans. GM-R-22 strain demonstrated the highest pectinolytic activity (0.751 U/mL) at pH 3.5 and 12 °C. Yeast pectinases were constitutively produced. This study is the first report about strains of A. pullulans producing pectinases which are able to show good activity at low temperature. These pectinolytic strains could be of interest in wine production.
Asunto(s)
Poligalacturonasa/aislamiento & purificación , Vino/microbiología , Levaduras/enzimología , Levaduras/aislamiento & purificación , Argentina , Frío , Ecosistema , Saccharomyces cerevisiae , Vitis/citología , Vitis/microbiologíaRESUMEN
Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 degrees C and had optimum activity at pH 5 and 50 degrees C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.
Asunto(s)
Coriolaceae/enzimología , Proteínas Fúngicas/aislamiento & purificación , Poligalacturonasa/aislamiento & purificación , Fraccionamiento Celular/métodos , Coriolaceae/crecimiento & desarrollo , Fermentación , Concentración de Iones de Hidrógeno , Micología/métodos , Solventes , TemperaturaRESUMEN
Polygalacturonases are pectinolytic enzymes that catalyze the hydrolysis of the plant cell-wall pectin backbone. They are widely used in the food industry for juice extraction and clarification. Aspergillus giganteus produces one polygalacturonase (PG) on liquid Vogel medium with citrus pectin as the only carbon source. In specific applications, such as those used in the food and medicine industries, the PG must be free of substances that could affect the characteristics of the product and the process, such as color, flavor, toxicity, and inhibitors. We present here an efficient, simple, and inexpensive method for purifying the A. giganteus PG and describe the characteristics of the purified enzyme. Purified PG was obtained after two simple steps: (1) protein precipitation with 70% ammonium sulfate saturation and (2) anion-exchange chromatography on a DEAE-Sephadex A-50 column. The final enzyme solution retained 86.4% of its initial PG activity. The purified PG had a molecular weight of 69.7 kDa, exhibited maximal activity at pH 6.0 and 55-60 degrees C, and was stable in neutral and alkaline media. It had a half-life of 115, 18, and 6 min at 40, 50 and 55 degrees C, respectively. Purified PG showed its highest hydrolytic activity with low-esterified and nonesterified substrates, releasing monogalacturonic acid from substrate, indicating that it is an exopolygalacturonase. PG activity was enhanced in the presence of beta-mercaptoethanol, dithiothreitol, Co(2+), Mn(2+), Mg(2+), NH(4) (+), and Na(+) and was resistant to inhibition by Pb(2+).
Asunto(s)
Aspergillus/enzimología , Proteínas Fúngicas/aislamiento & purificación , Poligalacturonasa/aislamiento & purificación , Cromatografía por Intercambio Iónico , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Pectinas/metabolismo , Poligalacturonasa/biosíntesis , Poligalacturonasa/químicaRESUMEN
Polygalacturonase (PG) production by Fomes sclerodermeus using solid-state fermentation (SSF) was carried out. Maximal PG activity (26 U/gdw) was obtained between days 11 and 13 at the end of exponential growth. PG activity in the crude extract was more stable at pH 5-6 and 30 °C and had optimum activity at pH 5 and 50 °C. Optimal conditions for PG extraction were: one time extraction with Na2SO4 as solvent with 10 min. of agitation. In a scale-up system, PG activity per gram of dry substrate decreased about 60% compared with the activity obtained in an Erlenmeyer flask; however, high total PG activity was obtained.
Se estudió la producción de poligalacturonasa (PG) por Fomes sclerodermeus usando técnicas de fermentación en estado sólido. La actividad PG máxima (26 U/g ps) fue observada entre los días 11 y 13. La actividad PG en los extractos crudos fue más estable a pH 5-6 y 30 °C, con una actividad óptima a pH 5 y a 50 °C. Las condiciones óptimas para la extracción de PG se lograron con una única extracción empleando Na2SO4 como solvente, con 10 minutos de agitación. En el escalado del sistema, la actividad PG por gramo de peso seco de sustrato disminuyó cerca de 60% comparada con la obtenida en frascos Erlenmeyer, pero la actividad total fue mayor.
Asunto(s)
Coriolaceae/enzimología , Proteínas Fúngicas/aislamiento & purificación , Poligalacturonasa/aislamiento & purificación , Fraccionamiento Celular/métodos , Coriolaceae/crecimiento & desarrollo , Fermentación , Concentración de Iones de Hidrógeno , Micología/métodos , Solventes , TemperaturaRESUMEN
An extracellular polygalacturonase (PG) produced from Paecilomyces variotii was purified to homogeneity through two chromatography steps using DEAE-Fractogel and Sephadex G-100. The molecular weight of P. variotii PG was 77,300 Da by gel filtration and SDS-PAGE. PG had isoelectric point of 4.37 and optimum pH 4.0. PG was very stable from pH 3.0 to 6.0. The extent of hydrolysis of different pectins by the purified enzyme was decreased with an increase in the degree of esterification. PG had no activity toward non-pectic polysaccharides. The apparent K(m) and V(max) values for hydrolyzing sodium polypectate were 1.84 mg/mL and 432 micromol/min/mg, respectively. PG was found to have temperature optimum at 65 degrees Celsius and was totally stable at 45 degrees Celsius for 90 min. Half-life at 55 degrees Celsius was 50.6 min. Almost all the examined metal cations showed partial inhibitory effects under enzymatic activity, except for Na(+1), K(+1), and Co(+2) (1 mM) and Cu(+2) (1 and 10 mM).
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Espacio Extracelular/enzimología , Paecilomyces/enzimología , Poligalacturonasa/aislamiento & purificación , Poligalacturonasa/metabolismo , Cationes , Cromatografía en Capa Delgada , Electroforesis en Gel de Poliacrilamida , Estabilidad de Enzimas/efectos de los fármacos , Espacio Extracelular/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Hidrólisis/efectos de los fármacos , Punto Isoeléctrico , Cinética , Metales/farmacología , Peso Molecular , Paecilomyces/efectos de los fármacos , Pectinas/metabolismo , Especificidad por Sustrato/efectos de los fármacos , TemperaturaRESUMEN
The present work describes the purification and characterization of a novel extracellular polygalacturonase, PGase I, produced by Pycnoporus sanguineus when grown on citrus fruit pectin. This substrate gave enhanced enzyme production as compared to sucrose and lactose. PGase I is an exocellular enzyme releasing galacturonic acid as its principal hydrolysis product as determined by TLC and orcinol-sulphuric acid staining. Its capacity to hydrolyze digalacturonate identified PGase I as an exo-polygalacturonase. SDS-PAGE showed that PGase I is an N-glycosidated monomer. The enzyme has a molecular mass of 42kDa, optimum pH 4.8 and stability between pH 3.8 and 8.0. A temperature optimum was observed at 50-60 degrees C, with some enzyme activity retained up to 80 degrees C. Its activation energy was 5.352calmol(-1). PGase I showed a higher affinity towards PGA than citric pectin (Km=0.55+/-0.02 and 0.72+/-0.02mgml(-1), respectively). Consequently, PGase I is an exo-PGase, EC 3.2.1.82.