RESUMO
The Y509E mutant of ß-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g-1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization.
RESUMO
Hemicelluloses are the second most abundant polysaccharide in plant biomass, in which xylan is the main constituent. Aiming at the total degradation of xylan and the obtention of fermentable sugars, several enzymes acting synergistically are required, especially ß-xylosidases. In this study, ß-xylosidase from Geobacillus thermodenitrificans (GtXyl) was expressed in E. coli BL21 and characterized. The enzyme GtXyl has been grouped within the family of glycoside hydrolases 43 (GH43). Results showed that GtXyl obtained the highest activity at pH 5.0 and temperature of 60 °C. In the additive's tests, the enzyme remained stable in the presence of metal ions and EDTA, and showed high tolerance to xylose, with a relative activity of 55.4% at 400 mM. The enzyme also presented bifunctional activity of ß-xylosidase and α-l-arabinofuranosidase, with the highest activity on the substrate p-nitrophenyl-ß-d-xylopyranoside. The specific activity on p-nitrophenyl-ß-d-xylopyranoside was 18.33 U mg-1 and catalytic efficiency of 20.21 mM-1 s-1, which is comparable to other ß-xylosidases reported in the literature. Putting together, the GtXyl enzyme presented interesting biochemical characteristics that are desirable for the application in the enzymatic hydrolysis of plant biomass, such as activity at higher temperatures, high thermostability and stability to metal ions.
Assuntos
Xilose , Xilosidases , Xilose/química , Xilanos/metabolismo , Escherichia coli/metabolismo , Xilosidases/metabolismo , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por SubstratoRESUMO
Corn cobs (CCs) are abundant xylan-rich agricultural wastes. Here, we compared CCs XOS yields obtained via two different pretreatment routs, alkali and hydrothermal, using a set of recombinant endo- and exo-acting enzymes from GH10 and GH11 families, which have different restrictions for xylan substitutions. Furthermore, impacts of the pretreatments on chemical composition and physical structure of the CCs samples were evaluated. We demonstrated that alkali pretreatment route rendered 59 mg of XOS per gram of initial biomass, while an overall XOS yield of 115 mg/g was achieved via hydrothermal pretreatment using a combination of GH10 and GH11 enzymes. These results hold a promise of ecologically sustainable enzymatic valorization of CCs via "green" and sustainable XOS production.
Assuntos
Xilanos , Zea mays , Humanos , Agricultura , ÁlcalisRESUMO
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.
Assuntos
Xilosidases , Estabilidade Enzimática , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Xilanos , Xilosidases/genética , Xilosidases/metabolismoRESUMO
Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 ß-xylosidase from Geobacillus stearothermophilus with dual activity of ß-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original ß-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the K m value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.
Assuntos
Substituição de Aminoácidos , Proteínas de Bactérias/química , Reagentes de Ligações Cruzadas/química , Geobacillus stearothermophilus/enzimologia , Glutaral/química , Glicosídeo Hidrolases/química , Agregados Proteicos , Proteínas de Bactérias/genética , Geobacillus stearothermophilus/genética , Glicosídeo Hidrolases/genética , Mutação de Sentido IncorretoRESUMO
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.
RESUMO
Abstract Xylan degradation is an important step in different industries, such as in biorefinery for biomass hydrolysis. Talaromyces wortmannii is a known fungus due to second metabolite production but only few works showed the xylanolytic potential of this fungus. In this way, the aim of this study was to evaluate the production of xylanolytic enzymes from T. wortmannii DR49 on industrial agro wastes. Cultivation in shake flask showed highest xylanase titration (10.3 U/mL; 9.5 U/mL) for wheat bran (WB) and hydrothermal pretreated sugar cane bagasse (HB); in β-xylosidase production WB and xylose were the best carbon sources (0.57 U/mL; 0.34 U/mL) respectively. STR cultivation revealed that 29°C and pH 6.0 were the best conditions for xylanase (14.5 U/mL) and β-xylosidase (1.7 U/mL) production. T. wortmannii DR49 showed to be a potential candidate for xylanolytic enzymes production using agro wastes in bioreactors, which has never been previously reported in this fungus.
RESUMO
Demand for fungal xylanases in industrial biotechnological processes shows a clear increase worldwide, so there is an interest in adjusting the conditions of microbial xylanases production. In this study, the ability of the fungus Fusarium solani to produce extracellular xylanases with low cellulolytic activity was optimized by Box Wilson design. The best culture conditions were determined to obtain a crude enzyme preparation with significant xylanolytic activity and little cellulolytic activity. In most treatments, the xylanolytic activity was higher than the cellulolytic activity. A negative effect on the production of endoxylanases, ß-xylosidases and endocellulases was observed with the increasing of xylan concentration. Increasing the incubation time adversely affected the production of endocellulases and ß-xylosidases. According to the mathematical model and experimental tests, it is possible to produce endoxylanases with minimal endocellulase activity increasing incubation time and the concentration of ammonium sulfate. The optimal culture conditions to produce a greater amount of endoxylanases (10.65U/mg) and low endocellulases from F. solani were: 2.5% (w/v) xylan, 5.0, 2.0 and 0.4g/l, of yeast extract, ammonium sulfate and urea, respectively, with 120h of incubation.
Assuntos
Celulases , Endo-1,4-beta-Xilanases/biossíntese , Fermentação , Fusarium , Concentração de Íons de Hidrogênio , Microbiologia Industrial , Projetos de PesquisaRESUMO
Xylan, a component of plant cell walls, is composed of a backbone of ß-1,4-linked xylopyranosyl units with a number of substituents. The complete degradation of xylan requires the action of several enzymes, among them ß-xylosidase. The fungus Penicillium purpurogenum secretes a number of enzymes participating in the degradation of xylan. In this study, a ß-xylosidase from this fungus was expressed in Pichia pastoris, and characterized. This enzyme (Xyl2) is a member of glycoside hydrolase family 3; it consists of a sequence of 792 residues including a signal peptide of 20 residues, with a theoretical molecular mass for the mature protein of 84.2â¯KDa and an isoelectric point of 5.07. The highest identity with a characterized fungal enzyme, is with a ß-xylosidase from Aspergillus oryzae (70%). The optimal activity of Xyl2 is found at pH 2.0 and 28⯰C. The enzyme is most stable at pH 2.0 and conserves 40% of activity at 42⯰C (after 1h incubation). The kinetic parameters for p-nitrophenyl-ß-d-xylopyranoside are: KM 0.53 mM, kcat 1*107 s-1 and kcat/KM 1.9*1010â¯M-1â¯s-1. The enzyme is about 10% active on p-nitrophenyl-α-l-arabinofuranoside. Xyl2 exhibits a high hydrolytic activity on xylooligosaccharides; it liberates xylose from beechwood and birchwood glucuronoxylan and it acts synergistically with endoxylanases in the degradation of xylan. Its low pH optimum make this enzyme particularly useful in potential applications requiring a low pH such as increasing the flavor of wine.
Assuntos
Penicillium/enzimologia , Penicillium/genética , Xilosidases/genética , Xilosidases/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Pichia/genéticaRESUMO
Biomass hydrolysis constitutes a bottleneck for the biotransformation of lignocellulosic residues into bioethanol and high-value products. The efficient deconstruction of polysaccharides to fermentable sugars requires multiple enzymes acting concertedly. GH43 ß-xylosidases are among the most interesting enzymes involved in hemicellulose deconstruction into xylose. In this work, the structural and functional properties of ß-xylosidase EcXyl43 from Enterobacter sp. were thoroughly characterized. Molecular modeling suggested a 3D structure formed by a conserved N-terminal catalytic domain linked to an ancillary C-terminal domain. Both domains resulted essential for enzymatic activity, and the role of critical residues, from the catalytic and the ancillary modules, was confirmed by mutagenesis. EcXyl43 presented ß-xylosidase activity towards natural and artificial substrates while arabinofuranosidase activity was only detected on nitrophenyl α-L-arabinofuranoside (pNPA). It hydrolyzed xylobiose and purified xylooligosaccharides (XOS), up to degree of polymerization 6, with higher activity towards longer XOS. Low levels of activity on commercial xylan were also observed, mainly on the soluble fraction. The addition of EcXyl43 to GH10 and GH11 endoxylanases increased the release of xylose from xylan and pre-treated wheat straw. Additionally, EcXyl43 exhibited high efficiency and thermal stability under its optimal conditions (40 °C, pH 6.5), with a half-life of 58 h. Therefore, this enzyme could be a suitable additive for hemicellulases in long-term hydrolysis reactions. Because of its moderate inhibition by monomeric sugars but its high inhibition by ethanol, EcXyl43 could be particularly more useful in separate hydrolysis and fermentation (SHF) than in simultaneous saccharification and co-fermentation (SSCF) or consolidated bioprocessing (CBP).
Assuntos
Enterobacter/enzimologia , Xilosidases/química , Xilosidases/classificação , Sequência de Aminoácidos , Biomassa , Domínio Catalítico , Endo-1,4-beta-Xilanases/química , Fermentação , Hidrólise , Lignina/metabolismo , Modelos Moleculares , Mutação , Estabilidade Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Triticum/metabolismo , Xilosidases/biossíntese , Xilosidases/genéticaRESUMO
One full-length ß-xylosidase gene (hxylA) was identified from the Humicola grisea var. thermoidea genome and the cDNA was successfully expressed by Pichia pastoris SMD1168. An optimization of enzyme production was carried out, and methanol was found to be the most important parameter. The purified enzyme was characterized and showed the optimal conditions for the highest activity at pH 7.0 and 50°C, being thermostable by maintaining 41% of its activity after 12h incubated at 50°C. HXYLA is a bifunctional enzyme; it showed both ß-xylosidase and α-arabinfuranosidase activities. The Km and Vmax values were 1.3mM and 39.1U/mg, respectively, against 4-nitrophenyl ß-xylopyranoside. HXYLA showed a relatively strong tolerance to xylose with high Ki value of 603mM, with the xylose being a non-competitive inhibitor. HXYLA was successfully used simultaneously and sequentially with an endo-xylanase for analysis of synergism in the degradation of commercial xylans. Furthermore, commercial cellulases supplementation with HXYLA during sugarcane bagasse hydrolysis increased hydrolysis in 29%. HXYLA is distinguished from other ß-xylosidases by the attractive characteristics for industrial applications such as thermostability, high tolerance xylose and saccharification of biomass by convert xylan into fementable monosaccharides and improve cellulose hydrolysis.
Assuntos
Celulose/metabolismo , Proteínas Recombinantes/metabolismo , Saccharum/química , Xilose/farmacologia , Xilosidases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência , Especificidade por Substrato , Xilosidases/química , Xilosidases/genéticaRESUMO
ABSTRACT A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its β-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of β-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high β-xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of β-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce β-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of β-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the β-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.
Assuntos
Ascomicetos/enzimologia , Xilosidases/metabolismo , Fermentação , Polissacarídeos/metabolismo , Polissacarídeos/química , Especificidade por Substrato , Temperatura , Xilose/metabolismo , Biomassa , Zea mays/química , Ativação Enzimática , Concentração de Íons de Hidrogênio , HidróliseRESUMO
A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its -xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of -xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high -xylosidases activities 1003 U/mL. According our known, this study is the first to show so high levels of -xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce -xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60 °C showing better thermostability at 55 °C. The saccharification ability of -xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50 °C. Our data strongly indicated that the -xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.(AU)
Assuntos
Xilosidases/administração & dosagem , Xilosidases/análise , Eurotiales/fisiologiaRESUMO
A new strain of Thermomyces lanuginosus was isolated from the Atlantic Forest biome, and its ß-xylosidases optimization in response to agro-industrial residues was performed. Using statistical approach as a strategy for optimization, the induction of ß-xylosidases activity was evaluated in residual corn straw, and improved so that the optimum condition achieved high ß-xylosidases activities 1003U/mL. According our known, this study is the first to show so high levels of ß-xylosidases activities induction. In addition, the application of an experimental design with this microorganism to induce ß-xylosidases has not been reported until the present work. The optimal conditions for the crude enzyme extract were pH 5.5 and 60°C showing better thermostability at 55°C. The saccharification ability of ß-xylosidase in the presence of hemicellulose obtained from corn straw raw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50°C. Our data strongly indicated that the ß-xylosidases activities was not subjected to the effects of potential enzyme inhibitors often produced during fermentation process. These data suggest the application of this enzyme studied for saccharification of hemicellulose, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production.
Assuntos
Ascomicetos/enzimologia , Fermentação , Xilosidases/metabolismo , Biomassa , Ativação Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Polissacarídeos/química , Polissacarídeos/metabolismo , Especificidade por Substrato , Temperatura , Xilose/metabolismo , Zea mays/químicaRESUMO
KEY MESSAGE: The results obtained indicate that a ß-xylosidase gene may act as good indicator of chilling tolerance and provide new insights into the complex issue of peach fruit woolliness. The storage of peaches at low temperatures for prolonged periods can induce a form of chilling injury (CI) called woolliness, characterized by a lack of juiciness and a mealy texture. As this disorder has been associated with abnormal cell wall dismantling, the levels of 12 transcripts encoding proteins involved in cell wall metabolism were analysed in cultivars with contrasting susceptibility to this disorder selected from five melting flesh peach cultivars. The resistant ('Springlady') and susceptible ('Flordaking') cultivars displayed differences in the level of expression of some of the selected genes during fruit softening and in woolly versus non-woolly fruits. From these genes, the level of expression of PpXyl, which encodes for a putative ß-xylosidase, was the one that presented the highest correlation (negative) with the susceptibility to woolliness. PpXyl expression was also analysed in a cultivar ('Rojo 2') with intermediate susceptibility to woolliness, reinforcing the conclusion about the correlation of PpXyl expression to the presence of woolliness symptom. Moreover, the level of expression of PpXyl correlated to protein level detected by Western blot. Analyses of the promoter region of the PpXyl gene (1637 bp) isolated from the three cultivars showed no differences suggesting that cis-elements from other regions of the genome and/or trans elements could be responsible of the differential PpXyl expression patterns. Overall, the results obtained indicate that PpXyl may act as a good indicator of woolliness tolerance and that the regulation of expression of this gene in different cultivars does not depend on sequences upstream the coding sequence.
Assuntos
Parede Celular/genética , Frutas/genética , Prunus persica/genética , Temperatura Baixa , Eletroforese em Gel de Poliacrilamida , Armazenamento de Alimentos , Frutas/fisiologia , Prunus persica/fisiologia , Característica Quantitativa Herdável , Reação em Cadeia da Polimerase em Tempo RealRESUMO
Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified: Kluyveromyces marxianus, Aspergillus niger, Aspergillus sydowii and Aspergillus fumigatus, and the isolates were screened for the production of key enzymes in the saccharification of lignocellulosic material. Among them, three strains were selected as good producers of hemicellulolitic enzymes: A. niger (SBCM3), A. sydowii (SBCM7) and A. fumigatus (SBC4). The best ß-xylosidase producer was A. niger SBCM3 strain. This crude enzyme presented optimal activity at pH 3.5 and 55 °C (141 U/g). For ß-glucosidase and xylanase the best producer was A. fumigatus SBC4 strain, whose enzymes presented maximum activity at 60 °C and pH 3.5 (54 U/g) and 4.0 (573 U/g), respectively. All these crude enzymes presented stability around pH 3.0-8.0 and up to 60 °C, which can be very useful in industrial processes that work at high temperatures and low pHs. These enzymes also exhibited moderate tolerance to ethanol and the sugars glucose and xylose. These similar characteristics among these fungal crude enzymes suggest that they can be used synergistically in cocktails in future studies of biomass conversion with potential application in several biotechnological sectors.
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus niger/enzimologia , Celulose/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Kluyveromyces/enzimologia , Saccharum/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , Sequência de Bases , Biomassa , Brasil , DNA Fúngico/genética , DNA Intergênico/genética , Fermentação , Kluyveromyces/isolamento & purificação , Kluyveromyces/metabolismo , Lignina/metabolismo , Tipagem Molecular , Técnicas de Tipagem Micológica , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified:
Assuntos
Aspergillus fumigatus/enzimologia , /enzimologia , Celulose/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Kluyveromyces/enzimologia , Saccharum/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus niger/isolamento & purificação , /metabolismo , Sequência de Bases , Biomassa , Brasil , DNA Fúngico/genética , DNA Intergênico/genética , Fermentação , Kluyveromyces/isolamento & purificação , Kluyveromyces/metabolismo , Lignina/metabolismo , Tipagem Molecular , Técnicas de Tipagem Micológica , RNA Ribossômico/genéticaRESUMO
Nineteen fungi and seven yeast strains were isolated from sugarcane bagasse piles from an alcohol plant located at Brazilian Cerrado and identified up to species level on the basis of the gene sequencing of 5.8S-ITS and 26S ribosomal DNA regions. Four species were identified:
Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus niger/enzimologia , Celulose/metabolismo , /metabolismo , Kluyveromyces/enzimologia , Saccharum/microbiologia , Xilosidases/metabolismo , beta-Glucosidase/metabolismo , Aspergillus fumigatus/isolamento & purificação , Aspergillus fumigatus/metabolismo , Aspergillus niger/isolamento & purificação , Aspergillus niger/metabolismo , Sequência de Bases , Biomassa , Brasil , DNA Fúngico/genética , DNA Intergênico/genética , Fermentação , Kluyveromyces/isolamento & purificação , Kluyveromyces/metabolismo , Lignina/metabolismo , Tipagem Molecular , Técnicas de Tipagem Micológica , RNA Ribossômico/genética , Análise de Sequência de DNARESUMO
ß-Xylosidases (EC 3.2.1.37) catalyze the hydrolysis of short xylooligosaccharides into xylose, which is an essential step in the complete depolymerization of xylan, the major hemicellulosic polysaccharide of plant cell walls, and has great biotechnological relevance for the production of lignocellulose-based biofuels and the paper industry. In this study, a GH43 ß-xylosidase identified from the bacterium Bacillus licheniformis (BlXylA) was cloned into the the pET-28a bacterial expression vector, recombinantly overexpressed in Escherichia coli BL21(DE3) cells and purified to homogeneity by metal-affinity and size-exclusion chromatography. The protein was crystallized in the presence of the organic solvent 2-methyl-2,4-pentanediol and a single crystal diffracted to 2.49â Å resolution. The X-ray diffraction data were indexed in the monoclinic space group C2, with unit-cell parameters a = 152.82, b = 41.9, c = 71.79â Å, ß = 91.7°. Structural characterization of this enzyme will contribute to a better understanding of the structural requirements for xylooligosaccharide specificity within the GH43 family.
Assuntos
Bacillus/química , Proteínas de Bactérias/química , Proteínas Recombinantes de Fusão/química , Xilosidases/química , Sequência de Aminoácidos , Bacillus/enzimologia , Proteínas de Bactérias/genética , Clonagem Molecular , Cristalização , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Glicóis , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Alinhamento de Sequência , Solventes , Xilosidases/genéticaRESUMO
Xylan is an abundant plant cell wall polysaccharide and its reduction to xylose units for subsequent biotechnological applications requires a combination of distinct hemicellulases and auxiliary enzymes, mainly endo-xylanases and ß-xylosidases. In the present work, a bifunctional enzyme consisting of a GH11 endo-1,4-ß-xylanase fused to a GH43 ß-xylosidase, both from Bacillus subtilis, was designed taking into account the quaternary arrangement and accessibility to the substrate. The parental enzymes and the resulting chimera were successfully expressed in Escherichia coli, purified and characterized. Interestingly, the substrate cleavage rate was altered by the molecular fusion improving at least 3-fold the xylose production using specific substrates as beechwood xylan and hemicelluloses from pretreated biomass. Moreover, the chimeric enzyme showed higher thermotolerance with a positive shift of the optimum temperature from 35 to 50 °C for xylosidase activity. This improvement in the thermal stability was also observed by circular dichroism unfolding studies, which seems to be related to a gain of stability of the ß-xylosidase domain. These results demonstrate the superior functional and stability properties of the chimeric enzyme in comparison to individual parental domains, suggesting the molecular fusion as a promising strategy for enhancing enzyme cocktails aiming at lignocellulose hydrolysis.