RESUMEN
Laccase isoforms from basidiomycetes exhibit a superior redox potential compared to commercially available laccases obtained from ascomycete fungi, rendering them more reactive toward mono-substituted phenols and polyphenolic compounds. However, basidiomycetes present limitations for large-scale culture in liquid media, restraining the current availability of laccases from this fungal class. To advance laccase production from basidiomycetes, a newly designed 14-L low-shear aerated and agitated bioreactor provided enzyme titers up to 23.5 IU/mL from Trametes versicolor cultures. Produced enzymes underwent ultrafiltration and LC/MS-MS characterization, revealing the predominant production of only two out of the ten laccases predicted in the T. versicolor genome. Process simulation and economic analysis using SuperPro designer® suggested that T. versicolor laccase could be produced at US$ 3.60/kIU in a 200-L/batch enterprise with attractive economic parameters and a payback period of 1.7 years. The study indicates that new bioreactors with plain design help to produce low-cost enzymes from basidiomycetes.
Asunto(s)
Reactores Biológicos , Lacasa , Lacasa/metabolismo , Lacasa/biosíntesis , Trametes/enzimología , PolyporaceaeRESUMEN
Xylanase enzymes are useful to fractionate plant biomass, producing xylan, xylooligosaccharides (XOS), and antioxidant-derived XOS. In a biorefinery, pretreated biomass can be digested with xylanase prior to cellulose saccharification, enhancing the product portfolio in the process. With this vision, this study highlighted a wide range of new products attainable from alkaline-sulfite-pretreated sugarcane bagasse by treatments with endo-xylanase under controlled conditions. The developed process provided a crude extract corresponding to 29.7% (w/w) of pretreated sugarcane bagasse. The crude extract included a relatively polymeric glucuronoarabinoxylan fraction, DP2-DP6 xylooligosaccharides, and aromatic compounds. The enzymatically produced extract was fractionated with increasing ethanol concentrations [up to 90% (v/v)], providing precipitation of varied polymeric xylan fractions (48% (w/w) of the crude extract) with average molar masses ranging from 28 kDa to 3.6 kDa. The fraction soluble in 90% ethanol was subjected to adsorption on 4% (w/v) activated charcoal and eluted with an ethanol gradient from 10% to 70% (v/v), thus providing xylooligosaccharides and aromatic fractions. Most of the xylooligosaccharides (74% of the eluted sugars) were washed out in 10%-30% ethanol. DP2 and DP3 structures predominated in the 10% ethanol fraction, while DP5 structures were significantly enriched in the 30% ethanol fraction. Higher ethanol concentrations desorbed xylooligosaccharides associated with higher amounts of aromatic compounds. Total aromatics, phenolic structures, and p-hydroxycinnamates predominated in the fractions desorbed with 60% and 70% ethanol. The antioxidant activity of produced fractions correlated with their phenolic contents. Compiled results indicate that a wide variety of products can be prepared from pretreated biomass using xylanase-aided extraction procedures. Recovered fractions presented different features and specific application prospects. Beyond polymeric xylan with low lignin contamination, xylooligosaccharides or even lignin-carbohydrate complexes with antioxidant activity can be included in the biorefinery portfolio based on the currently developed fractionation studies.
RESUMEN
The ability of white-rot fungi to degrade polysaccharides in lignified plant cell walls makes them a suitable reservoir for CAZyme prospects. However, to date, CAZymes from these species are barely studied, which limits their use in the set of choices for biomass conversion in modern biorefineries. The current work joined secretome studies of two representative white-rot fungi, Phanerochaete chrysosporium and Trametes versicolor, with expression analysis of cellobiohydrolase (CBH) genes, and use of the secretomes to evaluate enzymatic conversion of simple and complex sugarcane-derived substrates. Avicel was used to induce secretion of high levels of CBHs in the extracellular medium. A total of 56 and 58 proteins were identified in cultures of P. chrysosporium and T. versicolor, respectively, with 78-86% of these proteins corresponding to plant cell wall degrading enzymes (cellulolytic, hemicellulolytic, pectinolytic, esterase, and auxiliary activity). CBHI predominated among the plant cell wall degrading enzymes, corresponding to 47 and 34% of the detected proteins in P. chrysosporium and T. versicolor, respectively, which confirms that Avicel is an efficient CBH inducer in white-rot fungi. The induction by Avicel of genes encoding CBHs (cel) was supported by high expression levels of cel7D and cel7C in P. chrysosporium and T. versicolor, respectively. Both white-rot fungi secretomes enabled hydrolysis experiments at 10 FPU/g substrate, despite the varied proportions of CBHs and other enzymes present in each case. When low recalcitrance sugarcane pith was used as a substrate, P. chrysosporium and T. versicolor secretomes performed similarly to Cellic® CTec2. However, the white-rot fungi secretomes were less efficient than Cellic® CTec2 during hydrolysis of more recalcitrant substrates, such as acid or alkaline sulfite-pretreated sugarcane bagasse, likely because Cellic® CTec2 contains an excess of CBHs compared with the white-rot fungi secretomes. General comparison of the white-rot fungi secretomes highlighted T. versicolor enzymes for providing high glucan conversions, even at lower proportion of CBHs, probably because the other enzymes present in this secretome and CBHs lacking carbohydrate-binding modules compensate for problems associated with unproductive binding to lignin.
RESUMEN
Sugarcane bagasses from three experimental sugarcane hybrids and a mill-reference sample were used to compare the efficiency and mode of action of acid and alkaline sulfite pretreatment processes. Varied chemical loads and reaction temperatures were used to prepare samples with distinguished characteristics regarding xylan and lignin removals, as well as sulfonation levels of residual lignins. The pretreatment with low sulfite loads (5%) under acidic conditions (pH 2) provided maximum glucose yield of 70% during enzymatic hydrolysis with cellulases (10 FPU/g) and ß-glucosidases (20 UI/g bagasse). In this case, glucan enzymatic conversion from pretreated materials was mostly associated with extensive xylan removal (70-100%) and partial delignification occurred during the pretreatment. The use of low sulfite loads under acidic conditions required pretreatment temperatures of 160°C. In contrast, at a lower pretreatment temperature (120°C), alkaline sulfite process achieved similar glucan digestibility, but required a higher sulfite load (7.5%). Residual xylans from acid pretreated materials were almost completely hydrolysed by commercial enzymes, contrasting with relatively lower xylan to xylose conversions observed in alkaline pretreated samples. Efficient xylan removal during acid sulfite pretreatment and during enzymatic digestion can be useful to enhance glucan accessibility and digestibility by cellulases. Alkaline sulfite process also provided substrates with high glucan digestibility, mainly associated with delignification and sulfonation of residual lignins. The results demonstrate that temperature, pH, and sulfite can be combined for reducing lignocellulose recalcitrance and achieve similar glucan conversion rates in the alkaline and acid sulfite pretreated bagasses. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:944-951, 2018.
Asunto(s)
Celulosa/química , Saccharum/química , Celulasa/metabolismo , Glucosa/química , Lignina/químicaRESUMEN
Efficient hydrolysis of holocellulose depends on a proper balance between cellulase (endoglucanase, exoglucanase, ß-glucosidase) and xylanase activities. The present study aimed to induce the production of cellulases and xylanases using liquid cultures (one, two, three, and four fungal strains on the same bioreactor) of wild strains of Trichoderma harzianum, Aspergillus niger, and Fusarium oxysporum. The strains were identified by amplification and analysis of the ITS rDNA region and the obtained sequences were deposited in Genbank. Enzymes (endoglucanase, exoglucansae, ß-glucosidase, and xylanase activities) and the profile of extracellular protein isoforms (SDS-PAGE) produced by different fungal combinations (N = 14) were analyzed by Pearson's correlation matrix and principal component analysis (PCA). According to our results, induction of endoglucanase (19.02%) and ß-glucosidase (6.35%) were obtained after 4 days when A. niger and F. oxysporum were cocultured. The combination of A. niger-T. harzianum produced higher endoglucanase in a shorter time than monocultures. On the contrary, when more than two strains were cultured in the same reactor, the relationships of competition were established, trending to diminish the amount of enzymes and the extracellular protein isoforms produced. The xylanase production was sensible to stress produced by mixed cultures, decreasing their activity. This is important when the aim is to produce cellulase-free xylanase. In addition, exoglucanase activity did not change in the combinations tested.
Asunto(s)
Ascomicetos/crecimiento & desarrollo , Ascomicetos/metabolismo , Reactores Biológicos/microbiología , Celulasas/biosíntesis , Técnicas de Cocultivo , Microbiología Industrial/métodos , Ascomicetos/enzimología , Ascomicetos/aislamiento & purificación , Aspergillus niger/enzimología , Aspergillus niger/crecimiento & desarrollo , Aspergillus niger/aislamiento & purificación , Aspergillus niger/metabolismo , Biomasa , Celulasas/metabolismo , Celulosa/metabolismo , Fermentación , Proteínas Fúngicas/biosíntesis , Proteínas Fúngicas/metabolismo , Fusarium/enzimología , Fusarium/crecimiento & desarrollo , Fusarium/aislamiento & purificación , Fusarium/metabolismo , Interacciones Microbianas/fisiología , Trichoderma/enzimología , Trichoderma/crecimiento & desarrollo , Trichoderma/aislamiento & purificación , Trichoderma/metabolismo , Xilosidasas/biosíntesis , Xilosidasas/metabolismoRESUMEN
Aqueous two-phase systems (ATPSs) composed by UCON (ethylene oxide/propylene oxide copolymer) and potassium phosphate salts were for the first time evaluated in the recovery of Peniophora cinerea laccase from complex fermented medium. The ATPSs were obtained by combining the random copolymer UCON with KH2PO4, potassium phosphate buffer pH 7 or K2HPO4. According to the results, protein partition occurred predominantly toward the saline phase (bottom phase) of the ATPSs, while some contaminants such as pigments partitioned mainly to the top phase. In preliminary tests, it was found that the salt with the lowest pH value (KH2PO4, pH 4.6) stimulated the enzyme activity, while the other salts (pH between 7.0 and 9.5) caused a strong inhibition. However, the salt inhibition was not observed in the equilibrium phases of the UCON-Potassium phosphate ATPSs. The laccase recovery was high for all the biphasic systems, but the highest value (134%) was obtained when using UCON combined with KH2PO4. When compared to conventional concentration and purification methods (lyophilization, ammonium sulfate precipitation, ultrafiltration, and ion exchange chromatography), ATPS was demonstrated to be an efficient alternative for P. cinerea laccase recovery from fermented medium.
Asunto(s)
Basidiomycota/enzimología , Compuestos Epoxi/química , Óxido de Etileno/química , Lacasa/aislamiento & purificación , Fosfatos/química , Compuestos de Potasio/química , Sales (Química)RESUMEN
Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.
Asunto(s)
Acremonium/metabolismo , Celulosa/metabolismo , Etanol/metabolismo , Fusarium/metabolismo , Glucosa/metabolismo , Saccharum/química , Xilosa/metabolismo , Zea mays/microbiología , Microbiología IndustrialRESUMEN
Sugar cane bagasse is recalcitrant to enzymatic digestion, which hinders the efficient conversion of its polysaccharides into fermentable sugars. Alkaline-sulfite pretreatment was used to overcome the sugar cane bagasse recalcitrance. Chemical and structural changes that occurred during the pretreatment were correlated with the efficiency of the enzymatic digestion of the polysaccharides. The first 30 min of pretreatment, which removed approximately half of the initial lignin and 30% of hemicellulose seemed responsible for a significant enhancement of the cellulose conversion level, which reached 64%. After the first 30 min of pretreatment, delignification increased slightly, and hemicellulose removal was not enhanced; however, acid groups continued to be introduced into the residual lignin. Water retention values were 145% to the untreated bagasse and 210% to the bagasse pretreated for 120 min and fiber widths increased from 10.4 to 30 µm, respectively. These changes were responsible for an additional increase in the efficiency of enzymatic hydrolysis of the cellulose, which reached 92% with the 120 min pretreated sample.
Asunto(s)
Celulasa/metabolismo , Celulosa/metabolismo , Saccharum/metabolismo , Sulfitos/metabolismo , beta-Glucosidasa/metabolismo , Celulosa/química , Hidrólisis , Saccharum/química , Sulfitos/químicaRESUMEN
Chemithermomechanical (CTM) processing was used to pretreat sugarcane bagasse with the aim of increasing cell wall accessibility to hydrolytic enzymes. Yields of the pretreated samples were in the range of 75-94%. Disk refining and alkaline-CTM and alkaline/sulfite-CTM pretreatments yielded pretreated materials with 21.7, 17.8, and 15.3% of lignin, respectively. Hemicellulose content was also decreased to some extent. Fibers of the pretreated materials presented some external fibrillation, fiber curling, increased swelling, and high water retention capacity. Cellulose conversion of the alkaline-CTM- and alkaline/sulfite-CTM-pretreated samples reached 50 and 85%, respectively, after 96 h of enzymatic hydrolysis. Two samples with low initial lignin content were also evaluated after the mildest alkaline-CTM pretreatment. One sample was a partially delignified mill-processed bagasse. The other was a sugarcane hybrid selected in a breeding program. Samples with lower initial lignin content were hydrolyzed considerably faster in the first 24 h of enzymatic digestion. For example, enzymatic hydrolysis of the sample with the lowest initial lignin content (14.2%) reached 64% cellulose conversion after only 24 h of hydrolysis when compared with the 30% observed for the mill-processed bagasse containing an initial lignin content of 24.4%.
Asunto(s)
Celulosa/metabolismo , Hidrolasas/metabolismo , Lignina/análisis , Saccharum , Pared Celular/metabolismo , Hidrólisis , CinéticaRESUMEN
Experiments based on a 2(3) central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar-alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.
Asunto(s)
Celulosa/química , Ácidos Sulfúricos/farmacología , Celulosa/análisis , Celulosa/metabolismo , Hidrólisis , Lignina/análisis , Polisacáridos/análisis , Saccharum/química , Temperatura , Xilosa/análisisRESUMEN
This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, ß-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.
Asunto(s)
Celulasas/metabolismo , Celulosa/metabolismo , Saccharum/metabolismo , Ácidos Sulfúricos/química , Celulasa/metabolismo , Celulasas/química , Celulosa/química , Conservación de los Recursos Energéticos , Conservación de los Recursos Naturales , Etanol/economía , Etanol/metabolismo , Hidrólisis , Polisorbatos/metabolismo , Saccharum/química , beta-Glucosidasa/metabolismoRESUMEN
Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 2(2) factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.
Asunto(s)
Celulosa/química , Endo-1,4-beta Xilanasas/metabolismo , Proteínas Fúngicas/metabolismo , Polisacáridos/metabolismo , Saccharum/química , Thermoascus/enzimología , Xilosa/metabolismo , Álcalis/química , Endo-1,4-beta Xilanasas/química , Proteínas Fúngicas/química , Hidrólisis , CinéticaRESUMEN
In this paper, we present the responses of the white-rot fungus Perenniporia medulla-panis to iron availability with regard to alterations in growth, expression of cellular proteins, Fe3+-reducing activity, and Fe3+ chelators production. Iron supplementation stimulated fungal growth but did not result in a significant increase in biomass production. Catechol and hydroxamate derivatives were produced mainly under iron deficiency, and their productions were repressed under iron supplementation conditions. Perenniporia medulla-panis showed several cellular proteins in the range of 10-90 kDa. Some of them showed negative iron-regulation. Iron-supplemented medium also repressed both cell surface and extracellular Fe3+-reducing activities; however, the highest cell surface activity was detected at the initial growth phase, whereas extracellular activity increased throughout the incubation period. No significant production of chelators and extracellular Fe3+-reducing activity were observed within the initial growth phase, suggesting that the reduction of Fe3+ to Fe2+ is performed by ferrireductases.