RESUMEN
The enzymatic conversion of lignocellulosic biomass to fermentable sugars is determined by the enzymatic activity of cellulases; consequently, improving enzymatic activity has attracted great interest in the scientific community. Cocktails of commercial cellulase often have low ß-glucosidase content, leading to the accumulation of cellobiose. This accumulation inhibits the activity of the cellulolytic complex and can be used to determine the enzymatic efficiency of commercial cellulase cocktails. Here, a novel codon optimized ß-glucosidase gene (B-glusy) from Trichoderma reesei QM6a was cloned and expressed in three strains of Escherichia coli (E. coli). The synthetic sequence containing an open reading frame (ORF) of 1491 bp was used to encode a polypeptide of 497 amino acid residues. The ß-glucosidase recombinant protein that was expressed (57 kDa of molecular weight) was purified by Ni agarose affinity chromatography and visualized by SDS-PAGE. The recombinant protein was better expressed in E. coli BL21 (DE3), and its enzymatic activity was higher at neutral pH and 30 °C (22.4 U/mg). Subsequently, the ß-glucosidase was immobilized using magnetite nano-support, after which it maintained >65% of its enzymatic activity from pH 6 to 10, and was more stable than the free enzyme above 40 °C. The maximum immobilization yield had enzyme activity of 97.2%. In conclusion, ß-glucosidase is efficiently expressed in the microbial strain E. coli BL21 (DE3) grown in a simplified culture medium.
Asunto(s)
Enzimas Inmovilizadas , Escherichia coli , Proteínas Fúngicas , Expresión Génica , Hypocreales/genética , Nanopartículas de Magnetita/química , beta-Glucosidasa , Estabilidad de Enzimas , Enzimas Inmovilizadas/biosíntesis , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/aislamiento & purificación , Hypocreales/enzimología , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/química , beta-Glucosidasa/genética , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
Vegetable oil-derived biodiesels have a major quality problem due to the presence of precipitates formed by steryl glucosides, which clog filters and injectors of diesel engines. An efficient, scalable, and cost-effective method to hydrolyze steryl glucosides using thermostable enzymes has been developed. Here, methods to discover, express in recombinant microorganisms and manufacture enzymes with SGase activity, as well as methods to treat biodiesel with such enzymes, and to measure the content of steryl glucosides in biodiesel samples are presented.
Asunto(s)
Glucósidos/química , Fitosteroles/química , beta-Glucosidasa/metabolismo , Biocombustibles/análisis , Clonación Molecular/métodos , Enzimas/química , Hidrólisis , Aceites de Plantas , beta-Glucosidasa/biosíntesisRESUMEN
The hydrodynamic environment in bioreactors affects the oxygen transfer rate and the shear conditions during microbial cultivations. Therefore, assessment of the effect of the hydrodynamic environment on cellular morphology can contribute to favoring the production of metabolites of interest. The aim of this work was to use image analysis in order to quantify the fragmentation of Aspergillus niger pellets in a conventional bioreactor operated using different impeller speeds, air flow rates, and impeller configurations including Rushton turbines and Elephant Ear impellers, with evaluation of the influence of the hydrodynamic environment on the production of cellulolytic enzymes. An empirical kinetic model was proposed to describe the dynamics of pellet fragmentation and quantify the shear conditions. The results showed that the agitation speed affected the dynamics of pellet fragmentation in two ways, by accelerating the damage process and by increasing the magnitude of the fragmentation. Both endoglucanase and ß-glucosidase production exhibited a linear relationship with the pellet fragmentation percentage, which was directly related to the shear conditions. Interestingly, ß-glucosidase production was favored under high shear conditions, while the highest endoglucanase production occurred under low shear conditions. These findings may be useful for defining suitable systems and operating conditions for the production of metabolites including enzymes in bioreactors, as well as defining conditions that favour a specific pre-determined enzyme cocktail.
Asunto(s)
Aspergillus niger/enzimología , Reactores Biológicos , Celulasa/biosíntesis , beta-Glucosidasa/biosíntesis , Fermentación , Hidrodinámica , CinéticaRESUMEN
The use of plant biomass for biofuel production will require efficient utilization of the sugars in lignocellulose, primarily cellobiose, because it is the major soluble by-product of cellulose and acts as a strong inhibitor, especially for cellobiohydrolase, which plays a key role in cellulose hydrolysis. Commonly used ethanologenic yeast Saccharomyces cerevisiae is unable to utilize cellobiose; accordingly, genetic engineering efforts have been made to transfer ß-glucosidase genes enabling cellobiose utilization. Nonetheless, laboratory yeast strains have been employed for most of this research, and such strains may be difficult to use in industrial processes because of their generally weaker resistance to stressors and worse fermenting abilities. The purpose of this study was to engineer industrial yeast strains to ferment cellobiose after stable integration of tabgl1 gene that encodes a ß-glucosidase from Thermoascus aurantiacus (TaBgl1). The recombinant S. cerevisiae strains obtained in this study secrete TaBgl1, which can hydrolyze cellobiose and produce ethanol. This study clearly indicates that the extent of glycosylation of secreted TaBgl1 depends from the yeast strains used and is greatly influenced by carbon sources (cellobiose or glucose). The recombinant yeast strains showed high osmotolerance and resistance to various concentrations of ethanol and furfural and to high temperatures. Therefore, these yeast strains are suitable for ethanol production processes with saccharified lignocellulose.
Asunto(s)
Fermentación , Saccharomyces cerevisiae/genética , Thermoascus/enzimología , beta-Glucosidasa/biosíntesis , Biocombustibles , Biomasa , Ingeniería Genética , Microbiología Industrial , Lignina/metabolismo , Thermoascus/genética , beta-Glucosidasa/genéticaRESUMEN
Background: Cellulolytic enzymes of microbial origin have great industrial importance because of their wide application in various industrial sectors. Fungi are considered the most efficient producers of these enzymes. Bioprospecting survey to identify fungal sources of biomass-hydrolyzing enzymes from a high-diversity environment is an important approach to discover interesting strains for bioprocess uses. In this study, we evaluated the production of endoglucanase (CMCase) and ß-glucosidase, enzymes from the lignocellulolytic complex, produced by a native fungus. Penicillium sp. LMI01 was isolated from decaying plant material in the Amazon region, and its performance was compared with that of the standard isolate Trichoderma reesei QM9414 under submerged fermentation conditions. Results: The effectiveness of LMI01 was similar to that of QM9414 in volumetric enzyme activity (U/mL); however, the specific enzyme activity (U/mg) of the former was higher, corresponding to 24.170 U/mg of CMCase and 1.345 U/mg of ß-glucosidase. The enzymes produced by LMI01 had the following physicochemical properties: CMCase activity was optimal at pH 4.2 and the ß-glucosidase activity was optimal at pH 6.0. Both CMCase and ß-glucosidase had an optimum temperature at 60°C and were thermostable between 50 and 60°C. The electrophoretic profile of the proteins secreted by LMI01 indicated that this isolate produced at least two enzymes with CMCase activity, with approximate molecular masses of 50 and 35 kDa, and ß-glucosidases with molecular masses between 70 and 100 kDa. Conclusions: The effectiveness and characteristics of these enzymes indicate that LMI01 can be an alternative for the hydrolysis of lignocellulosic materials and should be tested in commercial formulations.
Asunto(s)
Penicillium/enzimología , Celulasa/biosíntesis , beta-Glucosidasa/biosíntesis , Oligosacáridos , Temperatura , Trichoderma/enzimología , Estabilidad de Enzimas , Celulasa/metabolismo , beta-Glucosidasa/metabolismo , Ecosistema Amazónico , Biocatálisis , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Lignina/metabolismoRESUMEN
The enhancement of enzyme complex produced by Penicillium echinulatum grown in several culture media components (bagasse sugarcane pretreated by various methods, soybean meal, wheat bran, sucrose, and yeast extract) was studied to increment FPase, xylanase, pectinase, and ß-glucosidase enzyme activities. The present results indicated that culture media composed with 10 g/L of the various bagasse pretreatment methods did not have any substantial influence with respect to the FPase, xylanase, and ß-glucosidase attained maximum values of, respectively, 2.68 FPU/mL, 2.04, and 115.4 IU/mL. On the other hand, proposed culture media to enhance ß-glucosidase production composed of 10 g/L steam-exploded bagasse supplemented with soybean flour 5.0 g/L, yeast extract 1.0 g/L, and sucrose 10.0 g/L attained, respectively, 3.19 FPU/mL and 3.06 IU/mL while xylanase was maintained at the same level. The proteomes obtained from the optimized culture media for enhanced FPase, xylanase, pectinase, and ß-glucosidase production were analyzed using mass spectrometry and a panel of GH enzyme activities against 16 different substrates. Culture medium designed to enhance ß-glucosidase activity achieved higher enzymatic activities values (13 measured activities), compared to the culture media for FPase/pectinase (9 measured activities) and xylanase (7 measured activities), when tested against the 16 substrates. Mass spectrometry analyses of secretome showed a consistent result and the greatest number of spectral counts of Cazy family enzymes was found in designed ß-glucosidase culture medium, followed by FPase/pectinase and xylanase. Most of the Cazy identified protein was cellobiohydrolase (GH6 and GH7), endoglucanase (GH5), and endo-1,4-ß-xylanase (GH10). Enzymatic hydrolysis of hydrothermally pretreated sugarcane bagasse performed with ß-glucosidase enhanced cocktail achieved 51.4 % glucose yield with 10 % w/v insoluble solids at enzyme load of 15 FPU/g material. Collectively the results demonstrated that it was possible to rationally modulate the GH activity of the enzymatic complex secreted by P. echinulatum using adjustment of the culture medium composition. The proposed strategy may contribute to increase enzymatic hydrolysis of lignocellulosic materials.
Asunto(s)
Medios de Cultivo/farmacología , Glicósido Hidrolasas/metabolismo , Complejos Multienzimáticos/metabolismo , Penicillium/efectos de los fármacos , Penicillium/enzimología , Celulasa/metabolismo , Celulosa , Medios de Cultivo/química , Medios de Cultivo/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Glicósido Hidrolasas/biosíntesis , Hidrólisis/efectos de los fármacos , Complejos Multienzimáticos/química , Penicillium/metabolismo , Poligalacturonasa/metabolismo , Saccharum , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/metabolismoRESUMEN
UNLABELLED: Coffee is among the most preferred nonalcoholic drinks, and its consumption is distributed globally. During the coffee fruiting process, however, a large amount of waste is generated in the form of pulp, mucilage, husks, and water waste. The pulp and mucilage have the chemical composition to support the growth of micro-organisms and the production of value-added product. The aim was testify pulp coffee can be considered as carbon and inductor source for ß-glucosidase by Bacillus subtilis CCMA 0087. The response surface methodology (RSM) based on a central composite rotatable design (CCRD) was employed for this optimization. The methodology used in the optimization process was validated by testing the best conditions obtained and comparing them with the values predicted by the model. The highest ß-glucosidase production (22·59 UI ml(-1) ) was reached in 24 h of culturing at coffee pulp concentration of 36·8 g l(-1) , temperature of 36·6°C, and pH of 3·64. SIGNIFICANCE AND IMPACT OF THE STUDY: Countries whose economy is based on agricultural activities generate a great deal of liquid and solid waste. Thus, it is important to develop new alternatives for using this waste rather than disposing it in the environment. The production of enzymes, and particularly cellulase, is one such alternative. In this study, we proposed to produce ß-glucosidase production from pulp coffee extract using a Bacillus subtilis strain.
Asunto(s)
Bacillus subtilis/metabolismo , Reactores Biológicos/microbiología , Café/metabolismo , Mucílago de Planta/metabolismo , beta-Glucosidasa/biosíntesis , Carbono , Celulasa/biosíntesis , Fermentación , Concentración de Iones de Hidrógeno , Temperatura , ResiduosRESUMEN
ß-Glucosidases are important enzymes with significant prospects in the industrial biotechnology, including their use in biomass hydrolysis for bioethanol production. In this study, the use of canola meal as carbon source for ß-glucosidase production by a Trichoderma viride strain in submerged fermentation was evaluated by applying central composite design and response surface methodology to optimize the production process. This statistical approach was also used to improve the passion fruit peel hydrolysis by T. viride crude extract. The model developed 3.6-fold increased ß-glucosidase activity. The culture conditions that resulted in the highest ß-glucosidase levels were a substrate concentration of 2.9 %, pH of medium 4.2 and cultivation time of 206 h. The ß-glucosidases produced under optimal conditions showed attractive properties for industrial applications, such as activity at high temperatures and stability at 55 °C and over a wide pH range. In addition, the enzymatic hydrolysis of passion fruit peel by T. viride crude extract was very promising, resulting in glucose yields of 66.4 %. This study, therefore, presents canola meal as an inexpensive and attractive substrate for the production of microbial ß-glucosidases.
Asunto(s)
Brassica rapa/microbiología , Residuos Industriales/prevención & control , Modelos Biológicos , Extractos Vegetales/metabolismo , Trichoderma/enzimología , beta-Glucosidasa/biosíntesis , Brassica rapa/química , Carbohidratos , Simulación por Computador , Activación Enzimática , Modelos Estadísticos , Especificidad por Sustrato , beta-Glucosidasa/aislamiento & purificaciónRESUMEN
AIMS: To obtain new cellulases and xylanases from thermophilic fungi; evaluate their potential for sugarcane bagasse saccharification. METHODS AND RESULTS: Thirty-two heat-tolerant fungi were isolated from the environment, identified (morphological/molecular tools) and the production of the enzymes was evaluated by solid state fermentation using lignocellulosic materials as substrates. Myceliophthora thermophila JCP 1-4 was the best producer of endoglucanase (357·51 U g(-1) ), ß-glucosidase (45·42 U g(-1) ), xylanase (931·11 U g(-1) ) and avicelase (3·58 U g(-1) ). These enzymes were most active at 55-70°C and stable at 30-60°C. Using crude enzymatic extract from M. thermophila JCP 1-4 to saccharify sugarcane bagasse pretreated with microwaves and glycerol, glucose and xylose yields obtained were 15·6 and 35·13% (2·2 and 1·95 g l(-1) ), respectively. CONCLUSIONS: All isolated fungi have potential to produce the enzymes; M. thermophila JCP 1-4 enzymatic extract have potential to be better explored in saccharification experiments. Pretreatment improved enzymatic saccharification, as sugar yields were much higher than those obtained from in natura bagasse. SIGNIFICANCE AND IMPACT OF THE STUDY: Myceliophthora thermophila JCP 1-4 produces avicelase (not commonly found among fungi; important to hydrolyse crystalline cellulose) and a ß-glucosidase resistant to glucose inhibition, interesting characteristics for saccharification experiments.
Asunto(s)
Celulasas/biosíntesis , Celulosa/metabolismo , Hongos/enzimología , Xilosidasas/biosíntesis , Ascomicetos/enzimología , Celulasa/biosíntesis , Celulasa/metabolismo , Celulasas/metabolismo , Fermentación , Hongos/aislamiento & purificación , Calor , Datos de Secuencia Molecular , Xilosidasas/metabolismo , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/metabolismoRESUMEN
Statistics based optimization, Plackett-Burman design (PBD) and response surface methodology (RSM) were employed to screen and optimize the media components for the production of naringinase from Aspergillus brasiliensis MTCC 1344, using solid state fermentation. Cassava waste (CW) was used as both the solid support and carbon source for the growth of A. brasiliensis. Based on the positive influence of the Pareto chart obtained from PBD on naringinase activity, three media components--maltose, peptone and calcium chloride were screened. Box-Behnken design (BBD) was employed using these three factors at three levels, for further optimization, and the second order polynomial equation was derived, based on the experimental data. Derringer's desired function methodology showed that the concentrations of maltose (7.74 g/L), peptone (4.19 g/L) and calcium chloride (7.63 mM) were the optimal levels for maximal naringinase activity (889.91 U/mg) which were validated through experiments.
Asunto(s)
Aspergillus/metabolismo , Medios de Cultivo , Fermentación , Complejos Multienzimáticos/biosíntesis , beta-Glucosidasa/biosíntesis , Algoritmos , Medios de Cultivo/química , Activación Enzimática , Modelos Estadísticos , Reproducibilidad de los ResultadosRESUMEN
BACKGROUND: In the last years, food grade antioxidants are used safely as an alternative to traditional fungicides to control fungal growth in several food and agricultural products. AIMS: In this work, the effect of butylated hydroxyanisole (BHA) and propyl paraben (PP) on two hydrolytic enzyme activity (ß-d-glucosidase and α-d-galactosidase) by Aspergillus section Nigri species under different water activity conditions (aW; 0.98, 0.95 and 0.93) and incubation time intervals (24, 48, 72 and 96h) was evaluated on peanut-based medium. METHODS: The activity of two glycosidases, ß-d-glucosidase and α-d-galactosidase, was assayed using as substrates 4-nitrophenyl-ß-d-glucopyranosido and 4-nitrophenyl-α-d-galactopyranosido, respectively. The enzyme activity was determined by the increase in optical density at 405nm caused by the liberation of p-nitrophenol by enzymatic hydrolysis of the substrate. Enzyme activity was expressed as micromoles of p-nitrophenol released per minute. RESULTS: The major inhibition in ß-d-glucosidase activity of A. carbonarius and A. niger was found with 20mmoll(-1) of BHA or PP at 0.98 and 0.95 aW, respectively, whereas for α-d-galactosidase activity a significant decrease in enzyme activity with respect to control was observed in A. carbonarius among 5 to 20mmoll(-1) of BHA or PP in all conditions assayed. Regarding A. niger, the highest percentages of enzyme inhibition activity were found with 20mmoll(-1) of BHA or PP at 0.95 aW and 96h. CONCLUSIONS: The results of this work provide information about the capacity of BHA and PP to inhibit in vitro conditions two of the most important hydrolytic enzymes produced by A. carbonarius and A. niger species.
Asunto(s)
Antioxidantes/farmacología , Aspergillus/efectos de los fármacos , Hidroxianisol Butilado/farmacología , Medios de Cultivo , Conservantes de Alimentos/farmacología , Proteínas Fúngicas/biosíntesis , Parabenos/farmacología , alfa-Galactosidasa/biosíntesis , beta-Glucosidasa/biosíntesis , Agar , Arachis , Aspergillus/enzimología , Aspergillus/crecimiento & desarrollo , Aspergillus niger/efectos de los fármacos , Aspergillus niger/enzimología , Aspergillus niger/crecimiento & desarrollo , Inducción Enzimática/efectos de los fármacos , Extractos Vegetales , Temperatura , Factores de Tiempo , AguaRESUMEN
Tapping causes the loss of large amounts of latex from laticifers and subsequently enhances latex regeneration, a high carbon- and nitrogen-cost activity in rubber tree. It is suggested that a 67 kDa protein associated with protein-storing cells in the inner bark tissues of rubber tree plays an important role in meeting the nitrogen demand for latex regeneration. Here, the 67 kDa protein was further characterized by a combination of cell biological, molecular biological and biochemical techniques. Immunogold labeling showed that the 67 kDa protein was specifically localized in the central vacuole of protein-storing cells. A full-length cDNA, referred to as HbVSP1, was cloned. The HbVSP1 contained a 1584 bp open reading frame encoding a protein of 527 amino acids. The putative protein HbVSP1 shared high identity with the P66 protein from rubber tree and proteins of the linamarase, and bg1A from cassava (Manihot esculenta). HbVSP1 contained the active site sequences of ß-glucosidase, TFNEP and I/VTENG. In vitro analysis showed that the 67 kDa protein exhibited the activity of both ß-glucosidase and linamarase and was thus characterized as a cyanogenic ß-glucosidase. Proteins immuno-related to the 67 kDa protein were present in leaves and lutoids of laticifers. Tapping down-regulated the expression of HbVSP1, but up-regulated the expression of genes encoding the key enzymes for rubber biosynthesis, while the effect of resting from tapping was the reverse. Taken together, the results suggest that the 67 kDa protein is a vacuole-localized cyanogenic ß-glucosidase encoded by HbVSP1 and may have a role in nitrogen storage in inner bark tissues of trunk during the leafless periods when rubber tree is rested from tapping.
Asunto(s)
Hevea/enzimología , Goma/metabolismo , beta-Glucosidasa/metabolismo , Secuencia de Aminoácidos , Regulación de la Expresión Génica de las Plantas , Hevea/genética , Datos de Secuencia Molecular , Corteza de la Planta/enzimología , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/genéticaRESUMEN
Humicola brevis var. thermoidea cultivated under solid state fermentation in wheat bran and water (1:2 w/v) was a good producer of ß-glucosidase and xylanase. After optimization using response surface methodology the level of xylanase reached 5,791.2 ± 411.2 U g(-1), while ß-glucosidase production was increased about 2.6-fold, reaching 20.7 ± 1.5 U g(-1). Cellulase levels were negligible. Biochemical characterization of H. brevis ß-glucosidase and xylanase activities showed that they were stable in a wide pH range. Optimum pH for ß-glucosidase and xylanase activities were 5.0 and 5.5, respectively, but the xylanase showed 80 % of maximal activity when assayed at pH 8.0. Both enzymes presented high thermal stability. The ß-glucosidase maintained about 95 % of its activity after 26 h in water at 55 °C, with half-lives of 15.7 h at 60 °C and 5.1 h at 65 °C. The presence of xylose during heat treatment at 65 °C protected ß-glucosidase against thermal inactivation. Xylanase maintained about 80 % of its activity after 200 h in water at 60 °C. Xylose stimulated ß-glucosidase activity up to 1.7-fold, at 200 mmol L(-1). The notable features of both xylanase and ß-glucosidase suggest that H. brevis crude culture extract may be useful to compose efficient enzymatic cocktails for lignocellulosic materials treatment or paper pulp biobleaching.
Asunto(s)
Ascomicetos/enzimología , Endo-1,4-beta Xilanasas/biosíntesis , beta-Glucosidasa/biosíntesis , Biomasa , Celulasa/aislamiento & purificación , Endo-1,4-beta Xilanasas/metabolismo , Estabilidad de Enzimas , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/metabolismo , Calor , Concentración de Iones de Hidrógeno , Cinética , Xilosa/farmacología , beta-Glucosidasa/metabolismoRESUMEN
The activity of ß-glucosidase (ßG), total cellulase (FPase) and endoglucanase (CMCase), produced by Aspergillus japonicus URM5620, was studied on solid-state fermentation using castor bean meal as substrate. The effect of the substrate amount, initial moisture, pH, and temperature on cellulase production was studied using a full factorial design (2(4)). The maximum ßG, FPase, and CMCase activity was 88.3, 953.4, and 191.6 U/g dry substrate, respectively. The best enzyme activities for all three enzymes were obtained at the same conditions with 5.0 g of substrate, initial moisture 15% at 25 °C and pH 6.0 with 120 h of fermentation. The optimum activity for FPase and CMCase was found at pH 3.0 at an optimum temperature of 50 °C for FPase and of 55 °C for CMCase. The cellulases were stable in the range of pH 3.0-10.0 at 50 °C temperature. The enzyme production optimization demonstrated clearly the impact of the process parameters on the yield of the cellulolytic enzymes.
Asunto(s)
Aspergillus/enzimología , Celulasa/biosíntesis , Microbiología Industrial/métodos , Ricinus communis/metabolismo , beta-Glucosidasa/biosíntesis , Algoritmos , Fermentación , Concentración de Iones de Hidrógeno , Cinética , Temperatura , Agua/metabolismoRESUMEN
beta-Glucosidase production by Debaryomyces pseudopolymorphus UCLM-NS7A using a simple nutrient medium containing cellobiose was evaluated under several biochemical and physiological parameters in submerged fermentation. Enzyme induction was also examined using different carbon and nitrogen sources. Cellobiose and ammonium nitrate were the best C and N sources to enhance beta-glucosidase production. The addition of NaCl, MgSO(4), yeast extract, ethanol and Tween 80 to the nutrient medium before inoculation was also compared. A factorial design to optimize enzyme production was developed using four variables that most influenced beta-glucosidase production and data analyzed by the response surface method. Optimal conditions to produce beta-glucosidase in shake-flasks were 1.25% cellobiose, 0.05% Tween 80, 0.4% NH(4)NO(3) over 72 hours. In another factorial design to further increase enzyme production, a lab fermenter using prior-determined shake-flask optimized conditions resulted in higher beta-glucosidase titres at 72 hours, pH controlled at 6.25 and agitation of 200 rpm.
Asunto(s)
Biotecnología/métodos , Espacio Extracelular/enzimología , Saccharomycetales/enzimología , beta-Glucosidasa/biosíntesis , Biomasa , Reactores Biológicos/microbiología , Fermentación , Concentración de Iones de Hidrógeno , Saccharomycetales/crecimiento & desarrollo , Propiedades de Superficie , Factores de TiempoRESUMEN
The production of cellulolytic enzymes by the fungus Aspergillus phoenicis was investigated. Grape waste from the winemaking industry was chosen as the growth substrate among several agro-industrial byproducts. A 2 x 2 central composite design was performed, utilizing the amount of grape waste and peptone as independent variables. The fungus was cultivated in submerged fermentation at 30 degrees C and 120 rpm for 120 h, and the activities of total cellulases, endoglucanases, and beta-glucosidases were measured. Total cellulases were positively influenced by the linear increase of peptone concentration and decrease at axial concentrations of grape waste and peptone. Maximum activity of endoglucanase was observed by a linear increase of both grape waste and peptone concentrations. Concentrations of grape waste between 5 and 15 g/L had a positive effect on the production of beta-glucosidase; peptone had no significant effects. The optimum production of the three cellulolytic activities was observed at values near the central point. A. phoenicis has the potential for the production of cellulases utilizing grape waste as the growth substrate.
Asunto(s)
Aspergillus/metabolismo , Celulasas/biosíntesis , Vitis/microbiología , Residuos , Celulasa/biosíntesis , Fermentación , beta-Glucosidasa/biosíntesisRESUMEN
Affinity chromatography with immobilised triazine dyes was used to separate the main enzymes present in the naringinase complex produced by Aspergillus terreus CECT 2663. One alpha-L-rhamnosidase and two beta-D-glucosidases (beta G1 and beta G2) were separated by a simple two-step procedure involving chromatography with Red HE-3B immobilised on Sepharose 4B first at pH 5.5 and then at pH 4.7. Maximum activity of the beta-D-glucosidases was from pH 4 to 6 and at 65 degrees C. Both glucosidases were active on p -nitrophenol glucoside and prunin with respective Km values of 1.9 mm and 1.6 mm for beta G1 and 2.1 mm and 0.25 mm for beta G2. Only beta G1 hydrolysed cellobiose (Km = 5.7 mm).
Asunto(s)
Aspergillus/enzimología , Fraccionamiento Químico/métodos , Cromatografía de Afinidad/métodos , Cromatografía en Agarosa/métodos , Glicósido Hidrolasas/biosíntesis , Glicósido Hidrolasas/aislamiento & purificación , Complejos Multienzimáticos/biosíntesis , Complejos Multienzimáticos/aislamiento & purificación , Triazinas , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/aislamiento & purificación , Adsorción , Colorantes , Activación Enzimática , Glicósido Hidrolasas/química , Concentración de Iones de Hidrógeno , Complejos Multienzimáticos/química , Temperatura , beta-Glucosidasa/químicaRESUMEN
The thermophilic fungus Chaetomium thermophilum var. coprophilum produced large amounts of extracellular and intracellular beta-glucosidase activity when grown on cellulose or cellobiose as carbon sources. The presence of glucose in the culture medium drastically decreased the level of beta-glucosidase activity, while cycloheximide prevented the induction of the extracellular enzyme activity by cellobiose. An extracellular beta-glucosidase induced by avicel was purified by a procedure involving acetone precipitation and chromatography on two DEAE-cellulose columns. The purified enzyme was a basic protein, with a carbohydrate content of 73%. The deglycosylated enzyme exhibited a molecular mass of 43 kDa, with pH and temperature optima of 5.5 and 65 degrees C respectively. The beta-glucosidase hydrolysed only cellobiose and p-nitrophenyl-beta-D-glucopyranoside, exhibiting apparent Km values of 3.13 mM and 0.76 mM, respectively. The native purified enzyme was stable up to 2 hours at 60 degrees C, and its thermal stability was directly dependent on glycosylation.
Asunto(s)
Chaetomium/enzimología , Calor , beta-Glucosidasa , Celobiosa/metabolismo , Celulosa/metabolismo , Chaetomium/crecimiento & desarrollo , Medios de Cultivo , Cicloheximida/farmacología , Estabilidad de Enzimas , Glucosa/metabolismo , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/química , beta-Glucosidasa/aislamiento & purificación , beta-Glucosidasa/metabolismoRESUMEN
The effect of carbon sources on the level of beta-1,3-glucanases in the culture filtrates of Trichoderma harzianum (Tc) was investigated. Enzyme activity was detected in all carbon sources, but highest levels were found when laminarin and purified cell walls were used. Three isoforms of beta-1,3-glucanase were produced during growth of the fungus on purified cell walls. Two isoforms were produced on chitin, chitosan, N-acetylglucosamine and laminarin, while only one was detected when the fungus was grown on cellulose and glucose. A 36-kDa beta-1,3-glucanase (GLU36) was secreted from T. harzianum (Tc) grown on all carbon sources tested as demonstrated by Western blot analysis. We found that a significant increase in the level of GLU36 in the culture filtrate follows glucose exhaustion, suggesting that this enzyme is controlled by carbon catabolite repression.
Asunto(s)
Regulación Fúngica de la Expresión Génica , Trichoderma/enzimología , beta-Glucosidasa/biosíntesis , beta-Glucosidasa/genética , Western Blotting , Carbono/metabolismo , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Glucano 1,3-beta-Glucosidasa , Trichoderma/genética , Trichoderma/crecimiento & desarrolloRESUMEN
Production of the components of the cellulase complex by Trametes trogii was investigated under different culture conditions. Endoglucanase, exoglucanase and beta-glucosidase were produced by Trametes trogii under shaken and stationary conditions using crystalline cellulose as the only carbon source. No marked differences were found for endo- and exoglucanase production between both culture conditions. In shaken cultures beta-glucosidase values doubled those obtained in stationary conditions. All maxima were reached approximately 10 days earlier in static cultures than in shaken ones. The addition of glucose to the culture medium improved growth but decreased abruptly the enzyme production. The optimal pH of the medium for the synthesis of the enzymes was 5.6, and the best incubation temperature was 25-28 degrees C. Enzyme activities were maximal at pH 3.1 and 45 degrees C (endoglucanase), pH 4.1 and 50 degrees C (exoglucanase) and pH 5.3 and 60 degrees C (beta-glucosidase).