RESUMEN
Xylooligosaccharides (XOS) are widely used in the food industry as prebiotic components. XOS with high purity are required for practical prebiotic function and other biological benefits, such as antioxidant and inflammatory properties. In this work, we immobilized the recombinant endo-1,4-ß-xylanase of Malbranchea pulchella (MpXyn10) in various chemical supports and evaluated its potential to produce xylooligosaccharides (XOS) from hydrothermal liquor of eucalyptus wood chips. Values >90% of immobilization yields were achieved from amino-activated supports for 120 min. The highest recovery values were found on Purolite (142%) and MANAE-MpXyn10 (137%) derivatives, which maintained more than 90% residual activity for 24 h at 70 °C, while the free-MpXyn10 maintained only 11%. In addition, active MpXyn10 derivatives were stable in the range of pH 4.0−6.0 and the presence of the furfural and HMF compounds. MpXyn10 derivatives were tested to produce XOS from xylan of various sources. Maximum values were observed for birchwood xylan at 8.6 mg mL−1 and wheat arabinoxylan at 8.9 mg mL−1, using Purolite-MpXyn10. Its derivative was also successfully applied in the hydrolysis of soluble xylan present in hydrothermal liquor, with 0.9 mg mL−1 of XOS after 3 h at 50 °C. This derivative maintained more than 80% XOS yield after six cycles of the assay. The results obtained provide a basis for the application of immobilized MpXyn10 to produce XOS with high purity and other high-value-added products in the lignocellulosic biorefinery field.
Asunto(s)
Eucalyptus , Xilanos , Madera , Glucuronatos , Oligosacáridos/química , Endo-1,4-beta Xilanasas , Prebióticos , HidrólisisRESUMEN
Since laccase acts specifically in lignin, the major contributor to biomass recalcitrance, this biocatalyst represents an important alternative to the pretreatment of lignocellulosic biomass. Therefore, this study investigates the laccase pretreatment and climate change effects on the hydrolytic performance of Panicum maximum. Through a Trop-T-FACE system, P. maximum grew under current (Control (C)) and future climate conditions: elevated temperature (2 °C more than the ambient canopy temperature) combined with elevated atmospheric CO2 concentration(600 µmol mol-1), name as eT+eC. Pretreatment using a laccase-rich crude extract from Lentinus sajor caju was optimized through statistical strategies, resulting in an increase in the sugar yield of P. maximum biomass (up to 57%) comparing to non-treated biomass and enabling hydrolysis at higher solid loading, achieving up to 26 g L-1. These increments are related to lignin removal (up to 46%) and lignin hydrophilization catalyzed by laccase. Results from SEM, CLSM, FTIR, and GC-MS supported the laccase-catalyzed lignin removal. Moreover, laccase mitigates climate effects, and no significant differences in hydrolytic potential were found between C and eT+eC groups. This study shows that crude laccase pretreatment is a potential and sustainable method for biorefinery solutions and helped establish P. maximum as a promising energy crop.
Asunto(s)
Lacasa/metabolismo , Lignina/química , Panicum/crecimiento & desarrollo , Biomasa , Carbohidratos , Cambio Climático , Hidrólisis/efectos de los fármacos , Lacasa/química , Lentinula , Lignina/metabolismo , AzúcaresRESUMEN
Increasing environmental and sustainability concerns, caused by current population growth, has promoted a raising utilization of renewable bio-resources for the production of materials and energy. Recently, nanocellulose (NC) has been receiving great attention due to its many attractive features such as non-toxic nature, biocompatibility, and biodegradability, associated with its mechanical properties and those related to its nanoscale, emerging as a promising material in many sectors, namely packaging, regenerative medicine, and electronics, among others. Nanofibers and nanocrystals, derived from cellulose sources, have been mainly produced by mechanical and chemical treatments; however, the use of cellulases to obtain NC attracted much attention due to their environmentally friendly character. This review presents an overview of general concepts in NC production. Especial emphasis is given to enzymatic hydrolysis processes using cellulases and the utilization of pulp and paper industry residues. Integrated process for the production of NC and other high-value products through enzymatic hydrolysis is also approached. Major challenges found in this context are discussed along with its properties, potential application, and future perspectives of the use of enzymatic hydrolysis as a pretreatment in the scale-up of NC production.
Asunto(s)
Celulasas/química , Celulosa/química , Nanofibras/química , Nanopartículas/química , Industria QuímicaRESUMEN
Cellulases constitute an enzymatic complex involved in the cellulose hydrolysis ß-1, 4-glycosidic linkages to release of glucose. Therefore, its application to degrade agro-industrial residues becomes relevant, since glucose is a product of industrial interest, aiming at its conversion into biocommodity production (e.g., enzymes, bioethanol and other value-added biochemicals). Thus, in natura Soybean hulls as well as fractions obtained from its alkaline, autohydrolysis and organosolv pretreatments were used as carbon sources in submerged fermentation processes to evaluate the cellulase-inducing capacity using a Penicillium sp. strain. Results showed an inductive effect on the production of 0.130 and 0.066 U/mL for CMCase and FPase, respectively, using 1% of the in natura residue. Regarding the fraction obtained from soybean hulls pretreated by autohydrolysis and organosolv, avicelase and ß-Glucosidase displayed a production of 0.200 and 0.550 U/mL, respectively. Therefore, the use of pretreated Soybean hull revealed its potential as an alternative carbon source for the cellulase production, which may contribute significantly to biotechnological purposes by adding value to an agro-industrial residue.
Asunto(s)
Celulasa/biosíntesis , Proteínas Fúngicas/biosíntesis , Glycine max/química , Penicillium/enzimología , Semillas/químicaRESUMEN
Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.
Asunto(s)
Enzimas Inmovilizadas/química , Hypocrea/química , Lipasa/química , Reactivos de Enlaces Cruzados/química , Bromuro de Cianógeno/química , Ácidos Docosahexaenoicos/química , Ácido Eicosapentaenoico/química , Activación Enzimática , Estabilidad de Enzimas , Glutaral/química , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Aceites/química , Desnaturalización Proteica , Estabilidad Proteica , Sefarosa/química , Solventes , Estereoisomerismo , Especificidad por Sustrato , TemperaturaRESUMEN
Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.
Asunto(s)
Aspergillus/enzimología , Glicósido Hidrolasas/metabolismo , Lignina/metabolismo , Proteínas Recombinantes/metabolismo , Reactores Biológicos/microbiología , Clonación Molecular , Activadores de Enzimas/análisis , Inhibidores Enzimáticos/análisis , Estabilidad de Enzimas , Fermentación , Expresión Génica , Glicósido Hidrolasas/química , Glicósido Hidrolasas/genética , Glicósido Hidrolasas/aislamiento & purificación , Concentración de Iones de Hidrógeno , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato , Tamarindus/química , TemperaturaRESUMEN
Xyloglucan-specific endo-ß-1,4-glucanases (Xegs, EC 3.2.1.151) exhibit high catalytic specificity for ß-1,4 linkages of xyloglucan, a branched hemicellulosic polysaccharide abundant in dicot primary cell walls and present in many monocot species. In nature, GH12 Xegs are not associated with carbohydrate-binding modules (CBMs), and here, we have investigated the effect of the fusion of the xyloglucan-specific CBM44 on the structure and function of a GH12 Xeg from Aspergillus niveus (XegA). This fusion presented enhanced catalytic properties and conferred superior thermal stability on the XegA. An increased k cat (chimera, 177.03 s(-1); XegA, 144.31 s(-1)) and reduced KM (chimera, 1.30 mg mL(-1); XegA, 1.50 mg mL(-1)) resulted in a 1.3-fold increase in catalytic efficiency of the chimera over the parental XegA. Although both parental and chimeric enzymes presented catalytic optima at pH 5.5 and 60 °C, the thermostabilitiy of the chimera at 60 °C was greater than the parental XegA. Moreover, the crystallographic structure of XegA together with small-angle X-ray scattering (SAXS) and molecular dynamics simulations revealed that the spatial arrangement of the domains in the chimeric enzyme resulted in the formation of an extended binding cleft that may explain the improved kinetic properties of the CBM44-XegA chimera.
Asunto(s)
Aspergillus/enzimología , Endo-1,3(4)-beta-Glucanasa/química , Endo-1,3(4)-beta-Glucanasa/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Glucanos/metabolismo , Xilanos/metabolismo , Secuencia de Aminoácidos , Aspergillus/química , Aspergillus/genética , Endo-1,3(4)-beta-Glucanasa/genética , Proteínas Fúngicas/genética , Glucanos/química , Cinética , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Ingeniería de Proteínas , Estructura Terciaria de Proteína , Dispersión del Ángulo Pequeño , Especificidad por Sustrato , Difracción de Rayos X , Xilanos/químicaRESUMEN
Daily agro-industrial waste, primarily cellulose, lignin, and hemicellulose, poses a significant environmental challenge. Harnessing lignocellulolytic enzymes, particularly endo-1,4-ß-xylanases, for efficient saccharification is a cost-effective strategy, transforming biomass into high-value products. This study focuses on the cloning, expression, site-directed mutagenesis, purification, three-dimensional modeling, and characterization of the recombinant endo-1,4-ß-xylanase (XlnA) from Aspergillus clavatus in Escherichia coli. This work includes evaluation of the stability at varied NaCl concentrations, determining kinetic constants, and presenting the heterologous expression of XlnAΔ36 using pET22b(+). The expression led to purified enzymes with robust stability across diverse pH levels, exceptional thermostability at 50 °C, and 96-100% relative stability after 24 h in 3.0 M NaCl. Three-dimensional modeling reveals a GH11 architecture with catalytic residues Glu 132 and 22. XlnAΔ36 demonstrates outstanding kinetic parameters compared to other endo-1,4-ß-xylanases, indicating its potential for industrial enzymatic cocktails, enhancing saccharification. Moreover, its ability to yield high-value compounds, such as sugars, suggests a promising and ecologically positive alternative for the food and biotechnology industries.
RESUMEN
The ß-glucosidase gene from Aspergillus nidulans FGSC A4 was cloned and overexpressed in the A. nidulans A773. The resulting purified ß-glucosidase, named AnGH3, is a monomeric enzyme with a molecular weight of approximately 80 kDa, as confirmed by SDS-PAGE. Circular dichroism further validated its unique canonical barrel fold (ß/α), a feature also observed in the 3D homology model of AnGH3. The most striking aspect of this recombinant enzyme is its robustness, as it retained 100% activity after 24 h of incubation at 45 and 50 ºC and pH 6.0. Even at 55 °C, it maintained 72% of its enzymatic activity after 6 h of incubation at the same pH. The kinetic parameters Vmax, KM, and Kcat/KM for ρ-nitrophenyl-ß-D-glucopyranoside (ρNPG) and cellobiose were also determined. Using ρNPG, the enzyme demonstrated a Vmax of 212 U mg - 1, KM of 0.0607 mmol L - 1, and Kcat/KM of 4521 mmol L - 1 s - 1 when incubated at pH 6.0 and 65 °C. The KM, Vmax, and Kcat/KM using cellobiose were 2.7 mmol L - 1, 57 U mg - 1, and 27 mmol -1 s - 1, respectively. AnGH3 activity was significantly enhanced by xylose and ethanol at concentrations up to 1.5 mol L - 1 and 25%, respectively. Even in challenging conditions, at 65 °C and pH 6.0, the enzyme maintained its activity, retaining 100% and 70% of its initial activity in the presence of 200 mmol L - 1 furfural and 5-hydroxymethylfurfural (HMF), respectively. The potential of this enzyme was further demonstrated by its application in the saccharification of the forage grass Panicum maximum, where it led to a 48% increase in glucose release after 24 h. These unique characteristics, including high catalytic performance, good thermal stability in hydrolysis temperature, and tolerance to elevated concentrations of ethanol, D-xylose, furfural, and HMF, position this recombinant enzyme as a promising tool in the hydrolysis of lignocellulosic biomass as part of an efficient multi-enzyme cocktail, thereby opening new avenues in the field of biotechnology and enzymology.
RESUMEN
Two bifunctional enzymes exhibiting combined xylanase and laccase activities were designed, constructed, and characterized by biochemical and biophysical methods. The Bacillus subtilis cotA and xynA genes were used as templates for gene fusion, and the xynA coding sequence was inserted into a surface loop of the cotA. A second chimera was built replacing the wild-type xynA gene by a thermostable variant (xynAG3) previously obtained by in vitro molecular evolution. Kinetic measurements demonstrated that the pH and temperature optima of the catalytic domains in the chimeras were altered by less than 0.5 pH units and 5 °C, respectively, when compared with the parental enzymes. In contrast, the catalytic efficiency (k(cat)/K(m)) of the laccase activity in both chimeras was 2-fold higher than for the parental laccase. Molecular dynamics simulations of the CotA-XynA chimera indicated that the two domains are in close contact, which was confirmed by the low resolution structure obtained by small angle x-ray scattering. The simulation also indicates that the formation of the inter-domain interface causes the dislocation of the loop comprising residues Leu-558 to Lys-573 in the laccase domain, resulting in a more accessible active site and exposing the type I Cu(2+) ion to the solvent. These structural changes are consistent with the results from UV-visible electronic and EPR spectroscopy experiments of the type I copper between the native and chimeric enzymes and are likely to contribute to the observed increase in catalytic turnover number.
Asunto(s)
Lacasa/metabolismo , Ingeniería de Proteínas/métodos , Proteínas Recombinantes de Fusión/metabolismo , Xilosidasas/metabolismo , Espectroscopía de Resonancia por Spin del Electrón , Concentración de Iones de Hidrógeno , Cinética , Lacasa/genética , Simulación de Dinámica Molecular , Proteínas Recombinantes de Fusión/genética , Xilosidasas/genéticaRESUMEN
Agroindustrial residues are materials often rich in cellulose and hemicellulose. The use of these substrates for the microbial production of enzymes of industrial interest is mainly due to their high availability associated with their low cost. In this work, corncob (CCs) particles decomposed to soluble compounds (liquor) were incorporated in the microbial growth medium through autohydrolysis, as a strategy to increase and undervalue xylanase and ß-xylosidase production by Aspergillus terricola and Aspergillus ochraceus. The CCs autohydrolysis liquor produced at 200 °C for 5, 15, 30 or 50 min was used as the sole carbon source or associated with untreated CC. The best condition for enzyme synthesis was observed with CCs submitted to 30 min of autohydrolysis. The enzymatic production with untreated CCs plus CC liquor was higher than with birchwood xylan for both microorganisms. A. terricola produced 750 total U of xylanase (144 h cultivation) and 30 total U of ß-xylosidase (96-168 h) with 0.75% untreated CCs and 6% CCs liquor, against 650 total U of xylanase and 2 total U of ß-xylosidase in xylan; A. ochraceus produced 605 total U of xylanase and 56 total U of ß-xylosidase (168 h cultivation) with 1% untreated CCs and 10% CCs liquor against 400 total U of xylanase and 38 total U of ß-xylosidase in xylan. These results indicate that the treatment of agroindustrial wastes through autohydrolysis can be a viable strategy in the production of high levels of xylanolytic enzymes.
Asunto(s)
Aspergillus/metabolismo , Endo-1,4-beta Xilanasas/biosíntesis , Xilosidasas/biosíntesis , Zea mays , Cromatografía Líquida de Alta Presión , Hidrólisis , Especificidad de la EspecieRESUMEN
BACKGROUND: Cellulose and hemicellulose are quantitatively the most important structural carbohydrates present in ruminant diets. Rumen micro-organisms produce enzymes that catalyse their hydrolysis, but the complex network formed by structural carbohydrates and lignin reduces their digestibility and restricts efficient utilisation of feeds by ruminants. This study aimed to produce two enzymatic extracts, apply them in ruminant diets to determine the best levels for ruminal digestibility and evaluate their effects on in vitro digestibility. RESULTS: In experiment 1 a two-stage in vitro technique was used to examine the effects of different enzymatic levels of Aspergillus japonicus and Aspergillus terricola on tropical forages. Enzyme addition had minor effects on corn silage at the highest enzymatic level. In experiment 2 an in vitro gas production (GP) technique was applied to determine apparent in vitro organic matter digestibility and metabolisable energy. The addition of enzymes in GP showed interesting results. Good data were obtained using sugar cane and Tifton-85 hay supplemented with extracts of A. japonicus and A. terricola respectively. CONCLUSION: Overall, the study suggests that addition of crude extracts containing exogenous fibrolytic enzymes to ruminant diets enhances the effective utilisation of ruminant feedstuffs such as forages.
Asunto(s)
Alimentación Animal , Aspergillus/enzimología , Productos Biológicos/farmacología , Dieta , Fibras de la Dieta/metabolismo , Digestión/efectos de los fármacos , Rumen/efectos de los fármacos , Animales , Celulosa/metabolismo , Fermentación , Gases/metabolismo , Poaceae , Polisacáridos/metabolismo , Rumen/microbiología , Rumen/fisiología , EnsilajeRESUMEN
Aspergillus ochraceus, a thermotolerant fungus isolated in Brazil from decomposing materials, produced an extracellular ß-xylosidase that was purified using DEAE-cellulose ion exchange chromatography, Sephadex G-100 and Biogel P-60 gel filtration. ß-xylosidase is a glycoprotein (39 % carbohydrate content) and has a molecular mass of 137 kDa by SDS-PAGE, with optimal temperature and pH at 70 °C and 3.0-5.5, respectively. ß-xylosidase was stable in acidic pH (3.0-6.0) and 70 °C for 1 h. The enzyme was activated by 5 mM MnCl2 (28 %) and MgCl2 (20 %) salts. The ß-xylosidase produced by A. ochraceus preferentially hydrolyzed p-nitrophenyl-ß-D-xylopyranoside, exhibiting apparent K(m) and V(max) values of 0.66 mM and 39 U (mg protein)⻹ respectively, and to a lesser extent p-nitrophenyl-ß-D-glucopyranoside. The enzyme was able to hydrolyze xylan from different sources, suggesting a novel ß-D-xylosidase that degrades xylan. HPLC analysis revealed xylans of different compositions which allowed explaining the differences in specificity observed by ß-xylosidase. TLC confirmed the capacity of the enzyme in hydrolyzing xylan and larger xylo-oligosaccharides, as xylopentaose.
Asunto(s)
Aspergillus ochraceus/enzimología , Xilanos/metabolismo , Xilosidasas/aislamiento & purificación , Xilosidasas/metabolismo , Aspergillus ochraceus/aislamiento & purificación , Brasil , Cloruros/metabolismo , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Microbiología Ambiental , Activadores de Enzimas/metabolismo , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Cinética , Cloruro de Magnesio/metabolismo , Compuestos de Manganeso/metabolismo , Peso Molecular , Especificidad por Sustrato , Temperatura , Xilosidasas/químicaRESUMEN
Fibrolytic enzyme production by Aspergillus japonicus C03 was optimized in a medium containing agro-industrial wastes, supplemented with peptone and yeast extract. A 2(3) full factorial composite and response surface methodology were used to design the experiments and analysis of results. Tropical forages were hydrolyzed by A. japonicus C03 enzymatic extract in different levels, and they were also tested as enzymatic substrate. Optimal production to xylanase was obtained with soybean bran added to crushed corncob (1:3), 0.01% peptone, and 0.2% yeast extract, initial pH 5.0, at 30 °C under static conditions for 5 days of incubation. Optimal endoglucanase production was obtained with wheat bran added to sugarcane bagasse (3:1), 0.01% peptone, and 0.2% yeast extract, initial pH 4.0, at 30 °C, for 6 days, under static conditions. Addition of nitrogen sources as ammonium salts either inhibited or did not influence xylanase production. This enzymatic extract had a good result on tropical forage hydrolyzes and showed better performance in the Brachiaria genera, due to their low cell wall lignin quantity. These results represent a step forward toward the use of low-cost agricultural residues for the production of valuable enzymes with potential application in animal feed, using fermentation conditions.
Asunto(s)
Alimentación Animal , Aspergillus/enzimología , Carbono/metabolismo , Celulasa/biosíntesis , Endo-1,4-beta Xilanasas/biosíntesis , Nitrógeno/metabolismo , Animales , Aspergillus/metabolismo , Brachiaria/química , Carbono/provisión & distribución , Celulasa/química , Cynodon/química , Endo-1,4-beta Xilanasas/química , Fermentación , Concentración de Iones de Hidrógeno , Hidrólisis , Nitrógeno/provisión & distribución , Panicum/química , Peptonas/metabolismo , Rumiantes , TemperaturaRESUMEN
The climate changes expected for the next decades will expose plants to increasing occurrences of combined abiotic stresses, including drought, higher temperatures, and elevated CO2 atmospheric concentrations. These abiotic stresses have significant consequences on photosynthesis and other plants' physiological processes and can lead to tolerance mechanisms that impact metabolism dynamics and limit plant productivity. Furthermore, due to the high carbohydrate content on the cell wall, plants represent a an essential source of lignocellulosic biomass for biofuels production. Thus, it is necessary to estimate their potential as feedstock for renewable energy production in future climate conditions since the synthesis of cell wall components seems to be affected by abiotic stresses. This review provides a brief overview of plant responses and the tolerance mechanisms applied in climate change scenarios that could impact its use as lignocellulosic biomass for bioenergy purposes. Important steps of biofuel production, which might influence the effects of climate change, besides biomass pretreatments and enzymatic biochemical conversions, are also discussed. We believe that this study may improve our understanding of the plant biological adaptations to combined abiotic stress and assist in the decision-making for selecting key agronomic crops that can be efficiently adapted to climate changes and applied in bioenergy production.
RESUMEN
This work presents an integrated and multi-step approach for the recovery and/or application of the lignocellulosic fractions from corncob in the production of high value added compounds as xylo-oligosaccharides, enzymes, fermentable sugars, and lignin in terms of biorefinery concept. For that, liquid hot water followed by enzymatic hydrolysis were used. Liquid hot water was performed using different residence times (10-50min) and holding temperature (180-200°C), corresponding to severities (log(R0)) of 3.36-4.64. The most severe conditions showed higher xylo-oligosaccharides extraction (maximum of 93%) into the hydrolysates and higher recovery of cellulose on pretreated solids (maximum of 65%). Subsequently, hydrolysates and solids were used in the production of xylanases and cellulases, respectively, as well as, pretreated solids were also subjected to enzymatic hydrolysis for the recovery of lignin and fermentable sugars from cellulose. Maximum glucose yield (100%) was achieved for solids pretreated at log(R0) of 4.42 and 5% solid loading.
Asunto(s)
Lignina , Biomasa , Celulosa , Hidrólisis , AzúcaresRESUMEN
Abstract We present a survey of projects that have been funded by FAPESP under the BIOTA-Microorganisms program. These projects generated a wide variety of results, including the identification of novel antibacterial-producing microorganisms, the characterization of novel microbial enzymes for industrial applications, taxonomic classification of novel microorganisms in several environments, investigation of the soil and mangrove microbial ecosystems and its influence on endangered plant species, and the sequencing of novel metagenome-assembled genomes. The results surveyed demonstrate the importance of microorganisms in environments that play important roles in human activities as well as the potential that many of these microorganisms have in contributing to biotechnological applications crucial for human survival in the 21st century.
Resumo Apresentamos um levantamento comentado de projetos financiados pelo programa BIOTA-Micro-organismos. Estes projetos geraram uma variada gama de resultados, incluindo a identificação de novos micro-organismos produtores de compostos antibacterianos, a caracterização de novas enzimas microbianas para usos industriais, classificação taxonômica de novos micro-organismos presentes em diversos ambientes, investigação de ecossistemas microbianos em solos e mangues e sua influência sobre plantas ameaçadas, e o sequenciamento de vários novos genomas microbianos derivados de metagenomas. Os resultados descritos demonstram o papel-chave de micro-organismos em ecossistemas importantes para atividades humanas, assim como o potencial que vários desses micro-organismos tem de contribuir para aplicações biotecnológicas cruciais para a sobrevivência humana no século 21.
RESUMEN
The trehalases from some thermophilic fungi, such as Humicola grisea, Scytalidium thermophilum, or Chaetomium thermophilum, possess mixed properties in comparison with those of the two main groups of trehalases: acid and neutral trehalases. Such as acid trehalases these enzymes are highly thermostable extracellular glycoproteins, which act at acidic pH. However, these enzymes are activated by calcium or manganese, and as a result inhibited by chelators and by ATP, properties typical of neutral trehalases. Here we extended the biochemical characterisation of these enzymes, by assaying their activity at acid and neutral pH. The acid activity (25-30% of total) was assayed in McIlvaine buffer at pH 4.5. Under these conditions the enzyme was neither activated by calcium nor inhibited by EDTA or ATP. The neutral activity was estimated in MES buffer at pH 6.5, after subtracting the activity resistant to EDTA inhibition. The neutral activity was activated by calcium and inhibited by ATP. On the other hand, the acid activity was more thermostable than the neutral activity, had a higher temperature optimum, exhibited a lower K(m), and different sensitivity to several ions and other substances. Apparently, these trehalases represent a new class of trehalases. More knowledge is needed about the molecular structure of this protein and its corresponding gene, to clarify the structural and evolutionary relationship of this trehalase to the conventional trehalases.
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Hongos/enzimología , Trehalasa/metabolismo , Adenosina Trifosfato/farmacología , Ácido Edético/farmacología , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Pronasa/farmacología , TemperaturaRESUMEN
The design of new biocatalysts through the immobilization of enzymes, improving their stability and reuse, plays a major role in the development of sustainable methodologies toward the so-called green chemistry. In this work, α-amylase (AAM) biocatalyst based on Mg3Al-layered double-hydroxide (LDH) matrix was successfully developed with the adsorption method. The adsorption process was studied and optimized as a function of time and enzyme concentration. The biocatalyst was characterized, and the mechanism of interaction between AAM and LDH, as well as the immobilization effects on the catalytic activity, was elucidated. The adsorption process was fast and irreversible, thus yielding a stable biohybrid material. The immobilized AAM partially retained its enzymatic activity, and the biocatalyst rapidly hydrolyzed starch in an aqueous solution with enhanced efficiency at intermediate loading values of ca. 50 mg/g of AAM/LDH. Multiple attachments through electrostatic interactions affected the conformation of the immobilized enzyme on the LDH surface. The biocatalyst was successfully stored in its dry form, retaining 100% of its catalytic activity. The results reveal the potential usefulness of a LDH compound as a support of α-amylase for the hydrolysis of starch that may be applied in industrial and pharmaceutical processes as a simple, environmentally friendly, and low-cost biocatalyst.
Asunto(s)
Hidróxidos/química , Nanoestructuras/química , Almidón/metabolismo , alfa-Amilasas/metabolismo , Aluminio/química , Biocatálisis , Electroforesis en Gel de Poliacrilamida , Enzimas Inmovilizadas/química , Enzimas Inmovilizadas/metabolismo , Hidrólisis , Magnesio/química , alfa-Amilasas/químicaRESUMEN
Production of multiple xylanases, in which each enzyme has a specific characteristic, can be one strategy to achieve the effective hydrolysis of xylan. Three xylanases (xyl 1, xyl 2, and xyl 3) from Aspergillus ochraceus were purified by chromatography using diethylaminoethyl (DEAE) cellulose, Biogel P-60, and Sephadex G-100 columns. These enzymes are glycoproteins of low molecular weight with an optimum temperature at 60 °C. The glycosylation presented is apparently not related to thermostability, since xyl 3 (20 % carbohydrate) was more thermostable than xyl 2 (67 % carbohydrate). Xyl 3 was able to retain most of its activity in a wide range of pH (3.5-8.0), while xyl 1 and xyl 2 presented optimum pH of 6.0. Xyl 1 and xyl 2 were activated by 5 and 10 mM MnCl2 and CoCl2, while xyl 3 was activated by 1 mM of the same compounds. Interestingly, xyl 2 presented high tolerance toward mercury ion. Xylanases from A. ochraceus hydrolyzed xylans of different origins, such as birchwood, oat spelt, larchwood, and eucalyptus (around 90 % or more), except xyl 2 and xyl 3 that hydrolyzed with lesser efficiency eucalyptus (66.7 %) and oat spelt (44.8 %) xylans.