RESUMEN

Corncob is an agro-residue rich in lignocellulosic material that can be used for the xylitol production, through its enzymatic conversion obtaining fermentable sugars and their subsequent fermentation. In light of the above, this study targeted the immobilization of Aspergillus labruscus xylanase and the use of the derivative to hydrolyze the corncob xylan for the obtainment of xylose, and its subsequent use for the production of xylitol. The extracellular xylanase was immobilized using different supports (sodium alginate, DEAE-Cellulose, DEAE-Sephadex and CM-Sephadex). Among all supports used, the best results were obtained with the DEAE-Cellulose derivative showing an efficiency of immobilization of 97-99%, yield of 93-95% and recovered activity of 81-100%. The sodium alginate derivative showed 3 cycles of reuse, with drop in activity of about 65% in the 3rd cycle using both CaCl2 and MnCl2 as crosslinkers. The best enzymatic activity for the DEAE-Cellulose derivative was observed at 55ºC and pH 5.0. This derivative presented reuse of 10 cycles using commercial xylan as substrate, and 4 cycles using corncob xylan. This derivative was used in an enzymatic reactor to hydrolyze corncob xylan, obtaining 2.7 mg/mL of xylose after 48 h of operation under optimal condition of temperature and pH. The xylose obtained from the corncob was fermented by Candida tropicalis for 96 h with consumption of 60%. The HPLC analyses indicated a production of 1.02 mg/mL of xylitol with 48 h of fermentation. In conclusion, this is the first report on the immobilization of the A. labrucus xylanase as an alternative for the obtainment of xylose from corncob xylan, and the subsequent production of xylitol.

RESUMEN

The development of technologies that allow the production of enzymes at a competitive cost is of great importance for several biotechnological applications, and the use of agro-industrial by-products is an excellent alternative to minimize costs and reduce environmental impacts. This study aimed to produce endo-xylanases using agro-industrial substrates rich in hemicellulose as sources of xylan in culture media. For this purpose, the yeast Cryptococcus laurentti and five lignocellulosic materials (defatted rice bran, rice husk, corn cob, oat husks, and soybean tegument), with and without pretreatment, were used as a source of xylan for enzyme production. To insert the by-products in the culture medium, they were dried and treated (if applicable) with 4% (w.v-1) NaOH and then added in a concentration of 2% (w.v-1). The cultures were agitated for 96 h, and the aliquots were removed to determine the enzymatic activities. Among the by-products studied, the maximum activity (8.7 U. mL-1 at pH 7.3) was obtained where rice bran was used. In contrast, corn cob was the by-product that resulted in lower enzyme production (1.6 U.mL-1). Thus, the defatted rice bran deserves special attention in front of the other by-products used since it provides the necessary substrate for producing endo-xylanases by yeast.

RESUMEN

BACKGROUND: Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fine industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. METHODS AND RESULTS: In this study, a gene encoding an esterase from Thermobifida fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purified using affinity chromatography. The TfEst kinetic assay revealed catalytic efficiencies of 0.58 s-1 mM-1, 1.09 s-1 mM-1, and 0.062 s-1 mM-1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/ß-hydrolase fold, which is consistent with other esterases. CONCLUSIONS: The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with significant implications for the advancement of biotechnology and biofuels industries.

Asunto(s)

Acetilesterasa , Esterasas , Thermobifida , Acetilesterasa/metabolismo , Acetilesterasa/genética , Acetilesterasa/química , Concentración de Iones de Hidrógeno , Cinética , Especificidad por Sustrato , Thermobifida/enzimología , Thermobifida/genética , Esterasas/metabolismo , Esterasas/genética , Esterasas/química , Estabilidad de Enzimas , Temperatura , Escherichia coli/genética , Escherichia coli/metabolismo , Clonación Molecular/métodos , Hidrólisis , Xilanos/metabolismo , Butiratos/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Nitrofenoles

RESUMEN

Hemicellulose plays a key role in both the production of cellulose nanofibrils (CNF) and their properties as suspensions and films. While the use of enzymatic and chemical pre-treatments for tailoring hemicellulose levels is well-established, post-treatment methods using enzymes remain relatively underexplored and hold significant promise for modifying CNF film properties. This study aimed to investigate the effects of enzymatic xylan removal on the properties of CNF film for packaging applications. The enzymatic post-treatment was carried out using an enzymatic cocktail enriched with endoxylanase (EX). The EX post-treated-CNFs were characterized by LALLS, XRD, and FEG-SEM, while their films were characterized in terms of physical, morphological, optical, thermal, mechanical, and barrier properties. Employing varying levels of EX facilitated the hydrolysis of 8 to 35 % of xylan, yielding CNFs with different xylan contents. Xylan was found to be vital for the stability of CNF suspensions, as its removal led to the agglomeration of nanofibrils. Nanostructures with preserved crystalline structures and different morphologies, including nanofibers, nanorods, and their hybrids were observed. The EX post-treatment contributed to a smoother film surface, improved thermostability, and better moisture barrier properties. However, as the xylan content decreased, the films became lighter (lower grammage), less strong, and more brittle. Thus, the enzymatic removal of xylan enabled the customization of CNF films' performance without affecting the inherent crystalline structure, resulting in materials with diverse functionalities that could be explored for use in packaging films.

Asunto(s)

Celulosa , Nanofibras , Xilanos , Xilanos/química , Nanofibras/química , Celulosa/química , Hidrólisis , Endo-1,4-beta Xilanasas/química , Endo-1,4-beta Xilanasas/metabolismo

RESUMEN

Agricultural and animal farming practices contribute significantly to greenhouse gas (GHG) emissions such as NH3, CH4, CO2, and NOx, causing local environmental concerns involving health risks and water/air pollution. A growing need to capture these pollutants is leading to the development of new strategies, including the use of solid adsorbents. However, commonly used adsorbent materials often pose toxicity and negative long-term environmental effects. This study aimed to develop responsive eco-friendly cryogels using xylan extracted from coffee parchment, a typical residue from coffee production. The crosslinking in cryogels was accomplished by "freeze-thawing" and subsequent freeze-drying. Cryogels were characterized in terms of morphology by using scanning electron microscopy, porosity, and density by the liquid saturation method and also moisture adsorption and ammonia adsorption capacity. The analysis showed that the porosity in the cryogels remained around 0.62-0.42, while the apparent densities varied from 0.14 g/cm3 to 0.25 g/cm3. The moisture adsorption capacity was the highest at the highest relative humidity level (80%), reaching 0.25-0.43 g of water per gram of sample; the amount of water adsorbed increased when the xylan content in the cryogel increased up to 10% w/v, which was consistent with the hygroscopic nature of xylan. The ammonia adsorption process was modeled accurately by a pseudo-second-order equation, where the maximum adsorption capacity in equilibrium reached 0.047 mg NH3/g when xylan reached 10% w/v in cryogels, indicating a chemisorption process. The cryogels under investigation hold promise for ammonia adsorption applications and GHG separation, offering a sustainable alternative for gas-capturing processes.

RESUMEN

Renewable materials of biological origin exhibit attractive properties in relation to traditional plastics, as they can be partially or completely replaced, thereby reducing environmental impacts. Hemicelluloses are a group of polysaccharides that have expanded applications when acetylated. Acetylation can improve the mechanical strength and water vapor barrier properties of xylan-based bioplastics. By partially acetylating xylan in the present study, it was possible to use water as a solvent for the film-forming solution and starch as a second polysaccharide in the formation of bioplastics. Xylan was modified via partial chemical acetylation by varying the reaction time, solvent, and catalyst content. The bioplastics were formed by non-acetylated xylan and acetylated xylan with degrees of substitution (DS) of 0.45 and 0.9, respectively, with starch to form blends using glycerol as a plasticizer. Acetylation with DS 0.45 showed better results in increasing the hydrophilicity of the bioplastic. On the other hand, acetylation influenced the thermal stability of bioplastics, increasing the maximum temperature of the degradation rate from 302 °C to 329 °C and 315 °C, owing to changes in the crystallinity of the polymers. In addition to the modulus of elasticity 2.99 to 290.61 and 274.67 MPa for the non-acetylated bioplastic and the bioplastic with DS of 0.45 and 0.90, respectively. Thus, the films obtained presented suitable physicochemical properties for use in various industrial applications, such as active and intelligent packaging in the food sector.

Asunto(s)

Almidón , Xilanos , Almidón/química , Xilanos/química , Vapor , Plásticos , Solventes

RESUMEN

There is an increasing need for renewable energy sources to replace part of our fossil fuel-based economy and reduce greenhouse gas emission. Sugarcane bagasse is a prominent feedstock to produce cellulosic bioethanol, but strategies are still needed to improve the cost-effective exploitation of this potential energy source. In model plants, it has been shown that GUX genes are involved in cell wall hemicellulose decoration, adding glucuronic acid substitutions on the xylan backbone. Mutation of GUX genes increases enzyme access to cell wall polysaccharides, reducing biomass recalcitrance in Arabidopsis thaliana. Here, we characterized the sugarcane GUX genes and silenced GUX2 in commercial hybrid sugarcane. The transgenic lines had no penalty in development under greenhouse conditions. The sugarcane GUX1 and GUX2 enzymes generated different patterns of xylan glucuronidation, suggesting they may differently influence the molecular interaction of xylan with cellulose and lignin. Studies using biomass without chemical or steam pretreatment showed that the cell wall polysaccharides, particularly xylan, were less recalcitrant in sugarcane with GUX2 silenced than in WT plants. Our findings suggest that manipulation of GUX in sugarcane can reduce the costs of second-generation ethanol production and enhance the contribution of biofuels to lowering the emission of greenhouse gases.

Asunto(s)

Arabidopsis , Saccharum , Celulosa/metabolismo , Xilanos/química , Biomasa , Polisacáridos , Arabidopsis/genética , Plantas/metabolismo

RESUMEN

Background Esterases (EC 3.1.1.X) are enzymes that catalyze the hydrolysis ester bonds. These enzymes have large potential for diverse applications in fne industries, particularly in pharmaceuticals, cosmetics, and bioethanol production. Methods and results In this study, a gene encoding an esterase from Thermobifda fusca YX (TfEst) was successfully cloned, and its product was overexpressed in Escherichia coli and purifed using afnity chromatography. The TfEst kinetic assay revealed catalytic efciencies of 0.58 s −1 mM−1, 1.09 s−1 mM−1, and 0.062 s−1 mM−1 against p-Nitrophenyl acetate, p-Nitrophenyl butyrate, and 1-naphthyl acetate substrates, respectively. Furthermore, TfEst also exhibited activity in a pH range from 6.0 to 10.0, with maximum activity at pH 8.0. The enzyme demonstrated a half-life of 20 min at 70 °C. Notably, TfEst displayed acetyl xylan esterase activity as evidenced by the acetylated xylan assay. The structural prediction of TfEst using AlphaFold indicated that has an α/β-hydrolase fold, which is consistent with other esterases. Conclusions The enzyme stability over a broad pH range and its activity at elevated temperatures make it an appealing candidate for industrial processes. Overall, TfEst emerges as a promising enzymatic tool with signifcant implications for the advancement of biotechnology and biofuels industries.

RESUMEN

This study aims to provide a comprehensive understanding of the key factors influencing the rheological behavior and the mechanisms of natural polyelectrolyte complexes (PECs) as flocculation agents for cellulose microfibers (CMFs) and nanofibers (CNFs). PECs were formed by combining two polyelectrolytes: xylan (Xyl) and chitosan (Ch), at different Xyl/Ch mass ratios: 60/40, 70/30, and 80/20. First, Xyl, Ch, and PEC solutions were characterized by measuring viscosity, critical concentration (c*), rheological parameter, ζ-potential, and hydrodynamic size. Then, the flocculation mechanisms of CMF and CNF suspensions with PECs under dynamic conditions were studied by measuring viscosity, while the flocculation under static conditions was examined through gel point measurements, floc average size determination, and ζ-potential analysis. The findings reveal that PEC solutions formed with a lower xylan mass ratio showed higher intrinsic viscosity, higher hydrodynamic size, higher z-potential, and a lower c*. This is due to the high molecular weight, charge, and gel-forming ability. All the analyzed solutions behave as a typical non-Newtonian shear-thinning fluid. The flocculation mechanisms under dynamic conditions showed that a very low dosage of PEC (between 2 and 6 mg PEC/g of fiber) was sufficient to produce flocculation. Under dynamic conditions, an increase in viscosity indicates flocculation at this low PEC dosage. Finally, under static conditions, maximum floc sizes were observed at the same PEC dosage where minimum gel points were reached. Higher PEC doses were required for CNF suspensions than for CMF suspensions.

RESUMEN

The intestinal microbiome is a community of anaerobic microorganisms whose activities significantly impact human health. Its composition can be modulated by consuming foods rich in dietary fiber, such as xylan, a complex polysaccharide that can be considered an emerging prebiotic. In this work, we evaluated how certain gut bacteria acted as primary degraders, fermenting dietary fibers, and releasing metabolites that other bacteria can further use. Different bacterial strains of Lactobacillus, Bifidobacterium, and Bacteroides were evaluated for their ability to consume xylan and interact with one another. Results from unidirectional assays gave indications of possible cross-feeding between bacteria using xylan as a carbon source. Bidirectional assays showed that Bifidobacterium longum PT4 increased its growth in the presence of Bacteroides ovatus HM222. Proteomic analyses indicated that B. ovatus HM222 synthesizes enzymes facilitating xylan degradation, such as ß-xylanase, arabinosidase, L-arabinose isomerase, and xylosidase. Interestingly, the relative abundance of these proteins remains largely unaffected in the presence of Bifidobacterium longum PT4. In the presence of B. ovatus, B. longum PT4 increased the production of enzymes such as α-L-arabinosidase, L-arabinose isomerase, xylulose kinase, xylose isomerase, and sugar transporters. These results show an example of positive interaction between bacteria mediated by xylan consumption. Bacteroides degraded this substrate to release xylooligosaccharides, or monosaccharides (xylose, arabinose), which might support the growth of secondary degraders such as B. longum.

Asunto(s)

Bifidobacterium longum , Azúcares , Humanos , Xilanos , Proteómica , Bacteroides , Fibras de la Dieta

RESUMEN

Rahnella sp. ChDrAdgB13 is a dominant member of the gut bacterial core of species of the genus Dendroctonus, which is one of the most destructive pine forest bark beetles. The objectives of this study were identified in Rahnella sp. ChDrAdgB13 genome the glycosyl hydrolase families involved in carbohydrate metabolism and specifically, the genes that participate in xylan hydrolysis, to determine the functionality of a putative endo-1,4-ß-D-xylanase, which results to be bifunctional xylanase-ferulic acid esterase called R13 Fae and characterize it biochemically. The carbohydrate-active enzyme prediction revealed 25 glycoside hydrolases, 20 glycosyl transferases, carbohydrate esterases, two auxiliary activities, one polysaccharide lyase, and one carbohydrate-binding module (CBM). The R13 Fae predicted showed high identity to the putative esterases and glycosyl hydrolases from Rahnella species and some members of the Yersiniaceae family. The r13 fae gene encodes 393 amino acids (43.5 kDa), containing a signal peptide, esterase catalytic domain, and CBM48. The R13 Fae modeling showed a higher binding affinity to ferulic acid, α-naphthyl acetate, and arabinoxylan, and a low affinity to starch. The R13 Fae recombinant protein showed activity on α-naphthyl acetate and xylan, but not on starch. This enzyme showed mesophilic characteristics, displaying its optimal activity at pH 6.0 and 25°C. The enzyme was stable at pH from 4.5 to 9.0, retaining nearly 66-71% of its original activity. The half-life of the enzyme was 23 days at 25°C. The enzyme was stable in the presence of metallic ions, except for Hg2+. The products of R13 Fae mediated hydrolysis of beechwood xylan were xylobiose and xylose, manifesting an exo-activity. The results suggest that Rahnella sp. ChDrAdgB13 hydrolyze xylan and its products could be assimilated by its host and other gut microbes as a nutritional source, demonstrating their functional role in the bacterial-insect interaction contributing to their fitness, development, and survival.

RESUMEN

Tropical agriculture produces large amounts of lignocellulosic residues that can potentially be used as a natural source of value-added products. The complexity of lignocellulose makes industrial-scale processing difficult. New processing techniques must be developed to improve the yield and avoid this valuable resource going to waste. Hemicelluloses comprise a variety of polysaccharides with different backbone compositions and decorations (such as methylations and acetylations), and form part of an intricate framework that confers structural stability to the plant cell wall. Organisms that are able to degrade these biopolymers include earthworms (Eisenia fetida), which can rapidly decompose a wide variety of lignocellulosic substrates. This ability probably derives from enzymes and symbiotic microorganisms in the earthworm gut. In this work, two substrates with similar C/N ratios but different hemicellulose content were selected. Palm fibre and coffee husk have relatively high (28%) and low (5%) hemicellulose contents, respectively. A vermicomposting mixture was prepared for the earthworms to feed on by mixing a hemicellulose substrate with organic market waste. Xylanase activity was determined in earthworm gut and used as a selection criterion for the isolation of hemicellulose-degrading bacteria. Xylanase activity was similar for both substrates, even though their physicochemical properties principally pH and electrical conductivity, as shown by the MANOVA analysis) were different for the total duration of the experiment (120 days). Xylanolytic strains isolated from earthworm gut were identified by sequence analysis of the 16S rRNA gene. Our results indicate that the four Actinobacteria, two Proteobacteria, and one Firmicutes isolated are active participants of the xylanolytic degradation by microbiota in the intestine of E. fetida. Most bacteria were more active at pH 7 and 28 °C, and those with higher activities are reported as being facultatively anaerobic, coinciding with the microenvironment reported for the earthworm gut. Each strain had a different degradative capacity.

Asunto(s)

Oligoquetos , Animales , Bacterias/genética , Humanos , Intestinos , ARN Ribosómico 16S , Suelo

RESUMEN

Xylose is an abundant bioresource for obtaining diverse chemicals and added-value products. The production of xylose from green alternatives like enzymatic hydrolysis is an important step in a biorefinery context. This research evaluated the synergism among four classes of hydrolytic purified enzymes-endo-1,4-ß-xylanase, α-L-arabinofuranosidase, ß-xylosidase, and α-D-glucuronidase-over hydrolysis of glucuronoarabinoxylan (GAX) obtained from brewers' spent grain (BSG) after alkaline extraction and ethanol precipitation. First, monosaccharides, uronic acids and glycosidic-linkages of alkaline extracted GAX fraction from BSG were characterized, after that different strategies based on the addition of one or two families of enzymes-endo-1,4-ß-xylanase (GH10 and GH11) and α-L-arabinofuranosidase (GH43 and GH51)-cooperating with one ß-xylosidase (GH43) and one α-D-glucuronidase (GH67) into enzymatic hydrolysis were assessed to obtain the best yield of xylose. The xylose release was monitored over time in the first 90 min and after a prolonged reaction up to 48 h of reaction. The highest yield of xylose was 63.6% (48 h, 40 â„ƒ, pH 5.5), using a mixture of all enzymes devoid of α-L-arabinofuranosidase (GH43) family. These results highlight the importance of GH51 arabinofuranosidase debranching enzyme to allow a higher cleavage of the xylan backbone of GAX from BSG and their synergy with 2 endo-1,4-ß-xylanase (GH10 and GH11), one ß-xylosidase (GH43) and the inclusion of one α-D-glucuronidase (GH67) in the reaction system. Therefore, this study provides an environmentally friendly process to produce xylose from BSG through utilization of enzymes as catalysts.

RESUMEN

A busca por custo x benefício na avicultura envolve compreender como melhorar o desempenho animal através da alimentação ao mesmo tempo em que haja diminuição dos custos. Tendo em vista o crescente aumento monetário da matéria prima da ração, várias pesquisas se voltam para novas fontes energéticas derivados de plantas, que contem quantidades significativas de fibras e agem como fatores antinutricionais, interferindo no desempenho zootécnico das aves. Os principais componentes das fibras, as beta glucanas e xilanas, podem ser quebradas e melhor aproveitadas quando no uso de enzimas exógenas chamadas de carboidrases, sendo a beta glucanase e xilase as respectivas enzimas que fazem a lise desses componentes. É necessário, portanto, melhor compreensão da forma de ação de cada enzima, assim como a fonte nutritiva das bases alimentares alternativas. Essa obra tem por objetivo abordar e revisar as duas principais fontes de fibras, beta glucanas e xilanas, e suas respectivas enzimas, beta glucanase e xilase.(AU)

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The pursuit for cost-benefit in poultry farming involves understanding how to improve animal performance through feeding while reducing costs. In view of the growing monetary increase in feed raw material, several researches are focused on new energy sources derived from plants, which contain significant amounts of fiber and act as anti-nutritional factors, interfering with the zootechnical performance of birds. The main components of fiber, beta glucans and xylans, can be broken down and better utilized when using exogenous enzymes called carbohydrases, with beta glucanase and xylase being the respective enzymes that lyse these components. It is therefore necessary to better understand the mode of action of each enzyme, as well as the nutritive source of alternative food bases. This work aims to address and review the two main sources of fiber, beta glucans and xylans, and their respective enzymes, beta glucanase and xylase.(AU)

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La búsqueda de la relación costo-beneficio en la avicultura implica comprender cómo mejorar el rendimiento animal a través de la alimentación y reducir los costos. Ante el creciente aumento monetario de la materia prima de los alimentos, varias investigaciones se enfocan en nuevas fuentes de energía derivadas de las plantas, que contienen cantidades significativas de fibra y actúan como factores antinutricionales, interfiriendo en el desempeño zootécnico de las aves. Los principales componentes de la fibra, los betaglucanos y los xilanos, se pueden descomponer y utilizar mejor cuando se usan enzimas exógenas llamadas carbohidrasas, siendo la betaglucanasa y la xilasa las enzimas respectivas que lisan estos componentes. Por lo tanto, es necesario comprender mejor el modo de acción de cada enzima, así como la fuente nutritiva de las bases alimenticias alternativas. Este trabajo tiene como objetivo abordar y revisar las dos principales fuentes de fibra, beta glucanos y xilanos, y sus respectivas enzimas, beta glucanasa y xilasa.(AU)

Asunto(s)

Animales , Aves de Corral/fisiología , Xilanos/análisis , beta-Glucanos/análisis , Fenómenos Fisiológicos Nutricionales de los Animales/fisiología , Alimentación Animal/análisis

RESUMEN

Attainment of a stable and highly active ß-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant ß-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. ß-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L-1 h-1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized ß-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.

Asunto(s)

Xilosidasas , Estabilidad de Enzimas , Enzimas Inmovilizadas/metabolismo , Concentración de Iones de Hidrógeno , Hidrólisis , Xilanos , Xilosidasas/genética , Xilosidasas/metabolismo

RESUMEN

Bamboo is a grass that has gained economic attention in the food industry as a source of dietary fiber, and the young bamboo culm may be an alternative to supply fibers to the market. The objective was to evaluate and characterize different portions (bottom, middle and top) of the young bamboo culm fibrous fractions from Dendrocalamus asper, Bambusa tuldoides and B. vulgaris regarding their color parameters, physicochemical composition and hemicellulosic polysaccharide characterization. Fibrous fractions were obtained after starch extraction and results showed high amounts of total dietary fiber (79-89%). The hemicellulosic polysaccharide contents (35.4-41.5%) demonstrated great potential for commercial extraction and so, we extracted them with alkali and fractionated regarding their solubility in cold-water. Insoluble polysaccharides (KP fractions) were obtained in higher yields (from 21.2% to 38.5%) than the soluble ones (KS fractions, yields from 2.3% to 5.2%). Monosaccharide composition showed mainly xylose and arabinose, with some minor amounts of mannose and galactose in some fractions. In a detailed NMR analysis, the presence of neutral xylans and arabinoxylans could be observed in all studied bamboo species, which can be used in food products and also in the production of xylooligosaccharides (XOS), biomaterials and biofuels.

Asunto(s)

Bambusa , Fibras de la Dieta , Industria de Alimentos , Polisacáridos , Almidón

RESUMEN

Two extracellular xylanases, denominated X2 and X3, were purified and characterized from the halotolerant bacterium Bacillus sp. Asc6BA isolated from "Salar de Ascotán" in the Atacama Desert. Xylanases were purified by anion exchange, cation exchange and size exclusion liquid chromatography. Xylanase X2 and X3 were purified ~ 690-fold and ~ 629-fold, respectively, compared to the concentrated extracellular fraction with a final specific activity of 169 and 154 u mg-1, respectively. Optimal conditions of pH and temperature of xylanolytic activity were 6.0 and 60 °C for X2 and 7.0 and 60 °C for X3. Half-life of X2 xylanase was 30 min at 50 °C, while X3 xylanase was remarkably more thermostable, retaining more than 70% of its activity after 32 h of incubation at 50 °C. X2 exhibited Km, Vmax and kcat values of 7.17 mg mL-1, 1.28 mM min-1 mg-1 and 425.33 s-1, respectively. X3 exhibited Km, Vmax and kcat values of 6.00 mg mL-1, 19.25 mM min-1 mg-1 and 82,515 s-1, respectively. In addition to their thermal stabilities, these enzymes were shown to be resistant to freeze-drying. These stability properties, in addition to the ability of these enzymes to be active in a wide range of temperatures and pHs, make these xylanases good candidates for industrial applications.

Asunto(s)

Bacillus/enzimología , Proteínas Bacterianas/metabolismo , Clima Desértico , Endo-1,4-beta Xilanasas/metabolismo , Tolerancia a la Sal , Bacillus/genética , Proteínas Bacterianas/genética , Chile , Endo-1,4-beta Xilanasas/genética , Estabilidad de Enzimas , Concentración de Iones de Hidrógeno , Especificidad por Sustrato , Temperatura

RESUMEN

Grass cell walls have hydroxycinnamic acids attached to arabinosyl residues of arabinoxylan (AX), and certain BAHD acyltransferases are involved in their addition. In this study, we characterized one of these BAHD genes in the cell wall of the model grass Setaria viridis. RNAi silenced lines of S. viridis (SvBAHD05) presented a decrease of up to 42% of ester-linked p-coumarate (pCA) and 50% of pCA-arabinofuranosyl, across three generations. Biomass from SvBAHD05 silenced plants exhibited up to 32% increase in biomass saccharification after acid pre-treatment, with no change in total lignin. Molecular dynamics simulations suggested that SvBAHD05 is a p-coumaroyl coenzyme A transferase (PAT) mainly involved in the addition of pCA to the arabinofuranosyl residues of AX in Setaria. Thus, our results provide evidence of p-coumaroylation of AX promoted by SvBAHD05 acyltransferase in the cell wall of the model grass S. viridis. Furthermore, SvBAHD05 is a promising biotechnological target to engineer crops for improved biomass digestibility for biofuels, biorefineries and animal feeding.

Asunto(s)

Aciltransferasas/metabolismo , Ácidos Cumáricos/metabolismo , Setaria (Planta)/metabolismo , Xilanos/metabolismo , Biomasa , Pared Celular/metabolismo , Genes de Plantas , Redes y Vías Metabólicas , Polisacáridos/metabolismo , Setaria (Planta)/enzimología , Setaria (Planta)/genética

RESUMEN

Hydrothermal processing is an interesting biorefinery technology for converting lignocellulosic biomass into biofuels and biocompounds. This process is based on the selective solubilization and depolymerization of hemicellulose fraction (xylan) and may be considered beneficial, due to the possibility of obtaining xylooligosaccharides (XOS) with a degree of polymerization (DP) suitable for prebiotic applications. This study evaluated the effect of pressure (2.5 and 10 MPa) in a kinetic study (30 min) of hydrothermal treatment (180 °C) to optimize the extraction of XOS from mango seed shell. Total reducing sugars (TRS) values were close to the maximum in 15 min showing a slower rate for both pressures after this time, but at 10 MPa the value was 20 % lower than at 2.5 MPa. Based on these results, a new extraction was performed at 2.5 MPa and 15 min, and the extracted XOS were quantified, yielding 393.44 mg XOS/g xylan. XOS with a degree of polymerization between X2-X6 corresponded to 82.24 mg/g and XOS with X > 6 (or soluble xylan) corresponded to 311.20 mg/g. A low amount of xylose (8.81 mg/g xylan) was released, resulting in a hemicellulose conversion of 40.2 %. In general, approximately 8.1 kg of total XOS was produced from 100 kg of dried mango seed shell (X2-X6-1.7 kg and X > 6-6.4 kg).

RESUMEN

Attainment of a stable and highly active β-xylosidase is of major importance for the efficient and cost-competitive hydrolysis of hemicellulose xylan, as well as for its industrial conversion into biofuels and biochemicals. Here, a recombinant β-xylosidase of the glycoside hydrolase family (GH43) from Bacillus subtilis was produced in Escherichia coli culture, purified, and subsequently immobilized on agarose and chitosan. Glutaraldehyde and glyoxyl groups were evaluated as activating agents to select the most efficient derivative. Multi-point immobilization on agarose led to an extraordinary thermal stability (half-lives 3604 and 164-fold higher than the free enzyme, at 50° and 35 °C, respectively). Even for chitosan activated with glutaraldehyde, a low-cost support, thermal stability of the immobilized enzyme was 326 and 12-fold higher than the free enzyme at 50° and 35°C, respectively. Immobilized enzymes showed no release of any subunit for the agarose-glyoxyl derivative, and only a few ones for the support activated with glutaraldehyde. Most remarkably, the enzyme kinetic behavior after immobilization increased up to 4-fold in relation to the free one. β-xylosidase, a tetrameric enzyme with four identical subunits, exists in equilibrium between the monomeric and oligomeric forms in solution. Depending on the pH of immobilization, the enzyme oligomerization can be favored, thus explaining the hyperactivation phenomenon. Both glyoxyl-agarose and chitosan-glutaraldehyde derivatives were used to catalyze corncob xylan hydrolysis, reaching 72 % conversion, representing a xylose productivity of around 20 g L−1 h−1. After ten 4h-cycles (pH 6.0, 35 °C), the xylan-to-xylose conversion remained approximately unchanged. Therefore, the immobilized β-xylosidases prepared in this work can be of great interest as biocatalysts in a biorefinery context.