Production of substrates for determination of xylanase activity / Produção de substratos para determinação da actividade da xilanase

The hemicelulose is a molecule heterogeneous and, therefore, its extraction from different biomass is a challenge. In this context this study proposed an hemicelulose extraction from sugarcane biomass using alkaline pretreatment to determination of xylanase activities and evaluation of affinity between substrates and enzymes. The results suggest that the xylan from sugarcane leaves showed greater affinity with the enzymes, but the xylan from bagasse was with more efficient and pure extraction.

Xylan solubilization and use as carbon source/inductor for microbial xylanase production / Solubilização de Xylan e utilização como fonte/indutor de carbono para a produção de xilanase microbiana

Xylanases are enzymes that hydrolyze the main chain of xylan, producing xylooligosaccharides and xylose. These enzymes are of great importance for the pharmaceutical industries and also for the production of biofuels. For the production of microbial xylanases, xylan substrate is used, which can be obtained from different biomass such as sugarcane residue. However, the problem when using commercial xylanases for xylanase induction is its high cost. In this context, this study sought to use a methodology for extracting xylan from sugarcane in alkaline medium quickly and inexpensively, and also the application of this xylan as substrate for xylanase production, using for both fungi Aspergillus nidulans, Aspergillus tubingensis and Aspergillus versicolor. The cultures in the presence of 1% xylan for these microorganisms were compared with cultures containing 1% wheat bran, an important substrate for xylanase production. The results indicate that xylan extracted by this methodology, besides presenting some amount of lignin, contributed to a higher production of xylanases for A. nidulans, and in A. tubingensis and A. versicolor the production was close to the when wheat bran was used as an inducer.

Evaluation of xylooligosaccharides effect on the growth of probiotic microorganisms / Avaliação do efeito de xilooligossacarídeos no crescimento de micro-organismos probióticos

Xylan is the hemicellulose with the highest incidence in agricultural and agroindustrial residues. Xylooligosaccharides (XOS) are xylose oligomers recognized for their prebiotic potential, in addition to other biological properties that promote health. XOS were produced through enzymatic hydrolysis and their prebiotic effect was evaluated. Mostly, XOS with a high degree of polymerization were produced. The microorganisms used were able to grow in the medium containing the XOS produced. 

Extraction of hemicellulose from sugarcane bagasse for xylooligosaccharides production

Hemicelulose extraída do bagaço de cana-de-açúcar foi hidrolisada por enzimas de Thermoascus aurantiacus, Trichoderma reesei e Aspergilus niger para obtenção de xilo-oligossacarídeos (XOs). A hemicelulose foi extraída com hidróxido de sódio na presença de antraquinona, sulfito de sódio ou peróxido de hidrogênio. O uso de antraquinona ou sulfito aumentou o rendimento de extração, porém a hemicelulose apresentou baixa solubilidade em água, propriedade inadequada para a hidrólise enzimática. A extração da hemicellulose com peróxido de hidrogênio em meio alcalino foi otimizada através de um planejamento fatorial completo 24 variando-se a concentração de H2O2 de 2 a 6% (m/v), tempo de reação de 4 a 16 h, temperatura de 20 a 60°C e presença ou não de 0,5% de sulfato de magnésio. No ponto central o rendimento de extração de hemicelulose foi de 94,5% com remoção de mais que 88% da lignina. Um rendimento de 86% de hemicelulose com baixo teor de lignina (5,9%) foi obtido em 6% de peróxido de hidrogênio por 4h a 20°C. Nessa condição a hemicelulose apresentou massa molar de 21.000 g/mol, composição aproximada de 81% xilose, 4% de arabinose, 4% de glicose e 3% de ácidos urônicos, alta solubilidade em água (90 % em massa) e coloração amarelo claro. As enzimas usadas na hidrólise dessa hemicelulose foram produzidas pelo cultivo dos fungos em meio sólido contento farelo de trigo. Em todos os extratos foi observada baixa atividade de endoglucanase e β-xilosidase e elevadas atividades de endo-β-1,4-xilanase. A máxima atividade de xilanase foi produzida por T. aurantiacus (1500 U/g), enquanto A. niger produziu 500 U/g e T. reesei 240 U/g, em 5 dias de cultivo. O perfil de produção de XOs com enzimas de T. aurantiacus e T. reesei foi semelhante, o principal produto foi xilobiose, seguido por xilose, xilotriose, xilotetraose e xilopentaose, sendo esses XOs de cadeia linear. A hidrólise da hemicelulose com enzimas de A. niger produziu exclusivamente xilose, consequência da presença de elevada atividade de β-xilosidase. A velocidade de conversão da hemicelulose em XOs com as enzimas de T. reesei foi maior no início da reação (6 h), diminuindo a partir de 24 h, período em que inicia a produção de xilose. A influência da concentração de substrato e carga de xilanase na conversão da hemicelulose em XOs foi avaliada através de um planejamento experimental 22 com face centrada. A condição otimizada da hidrólise (2,6% substrato e 60 U/g de endo-β-1,4-xilanase) com o extrato de T. aurantiacus resultou em 42% de conversão em XOs. A otimização da hidrólise da hemicelulose com o extrato de T. reesei resultou em uma conversão máxima de 20%, com ótimo de 3,8 % de substrato e 87,5 U/g de endo-β-1,4-xilanase. A eficiência da hidrólise com enzimas de T. aurantiacus foi maior que a obtida com alguns extratos comerciais testados neste trabalho. Além disso, apresentaram capacidade de degradar hemiceluloses de diferentes fontes: bétula e semente de aveia, com composições variadas. Diferenças na composição de açúcares e teor de lignina não interferiram na ação dessas enzimas. A hidrólise enzimática mostrou-se mais apropriada para a produção de XOs do que a auto-hidrólise, que gerou predominantemente xilose e houve formação de furfural. Apesar do curto tempo de reação, a produção de XOs foi menor e há necessidade de purificação para obtenção de um produto final com características desejáveis.Hemicellulose extracted from sugarcane bagasse was hydrolyzed by enzymes from Thermoascus aurantiacus, Trichoderma reesei and Aspergilus niger to cause the degradation of xylan to xylooligosaccharides (XOs). Hemicellulose was extracted with hydrogen peroxide in the presence of antraquinone, sodium sulphite or hydrogen peroxide. Hemicelluloses extracted with antraquinone or sulphite presented low solubility in water, which is not appropriated to enzymatic hydrolysis. To maximize the hemicellulose yields several extraction conditions were examined applying the 24 factorial design: H2O2 concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60°C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H2O2 treatment for 4 h and 20°C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. The molecular weight of hemicellulose was 21,000 g/mol with composition of aproximately 81% xylose, 4% arabinose, 4% glucose and 3% uronic acids, high water solubility (90 %). Enzymes for hemicellulose hydrolysis were produced by the fungi on wheat bran. Cellulases and hemicellulases were present in all extracts especially the endo-β-1,4-xylanase. The profile of production of XOs obtained on hydrolysis with enzymes from T. aurantiacus and T. reesei was similar, with the main product xylobiose, followed by xylose, xylotriose, xylotetraose and xylopentaose, and these XOs showed linear chain. The hydrolysis of hemicellulose with enzymes of A. niger produced exclusively xylose, a consequence of β-xylosidase content. The rate of conversion of hemicellulose in XOs with enzymes of T. reesei was higher at the beginning of the reaction (6 h), decreasing from 24 h, when starts the production of xylose. The influence of substrate concentration and loading of xylanase in conversion of hemicellulose to XOs was evaluated by an 22 full factorial design with centered face. Optimization of hydrolysis (2.6% substrate and 60 U/g endo-β-1,4-xylanase) with the extract of T. aurantiacus resulted in 42 % conversion XOs. The optimization with the extract of T. reesei resulted in a conversion of hemicellulose up to 20%, with optimal substrate 3.8% and 87.5 U/g endo-β-1,4-xylanase. The efficiency of hydrolysis by enzymes from T. aurantiacus was superior to commercial extracts, and showed ability to degrade hemicelluloses of different compositions (birchwood and oat spelt). The structural differences, such as branches and lignin content did not affect the action of these enzymes. The differences in the efficiency and extent of enzymatic hydrolysis by enzymes of these fungi might have occurred in function of differences in physicochemical properties and specific activity. The enzymatic hydrolysis was more appropriate for production of XOs than autohydrolysis, which generated predominantly xylose and formation of furfural. Despite of short reaction time, the production of XOs was low and purification is needed in order to obtain a final product with desirable characteristics

Extraction of hemicellulose from sugarcane bagasse for xylooligosaccharides production

Hemicelulose extraída do bagaço de cana-de-açúcar foi hidrolisada por enzimas de Thermoascus aurantiacus, Trichoderma reesei e Aspergilus niger para obtenção de xilo-oligossacarídeos (XOs). A hemicelulose foi extraída com hidróxido de sódio na presença de antraquinona, sulfito de sódio ou peróxido de hidrogênio. O uso de antraquinona ou sulfito aumentou o rendimento de extração, porém a hemicelulose apresentou baixa solubilidade em água, propriedade inadequada para a hidrólise enzimática. A extração da hemicellulose com peróxido de hidrogênio em meio alcalino foi otimizada através de um planejamento fatorial completo 24 variando-se a concentração de H2O2 de 2 a 6% (m/v), tempo de reação de 4 a 16 h, temperatura de 20 a 60°C e presença ou não de 0,5% de sulfato de magnésio. No ponto central o rendimento de extração de hemicelulose foi de 94,5% com remoção de mais que 88% da lignina. Um rendimento de 86% de hemicelulose com baixo teor de lignina (5,9%) foi obtido em 6% de peróxido de hidrogênio por 4h a 20°C. Nessa condição a hemicelulose apresentou massa molar de 21.000 g/mol, composição aproximada de 81% xilose, 4% de arabinose, 4% de glicose e 3% de ácidos urônicos, alta solubilidade em água (90 % em massa) e coloração amarelo claro. As enzimas usadas na hidrólise dessa hemicelulose foram produzidas pelo cultivo dos fungos em meio sólido contento farelo de trigo. Em todos os extratos foi observada baixa atividade de endoglucanase e β-xilosidase e elevadas atividades de endo-β-1,4-xilanase. A máxima atividade de xilanase foi produzida por T. aurantiacus (1500 U/g), enquanto A. niger produziu 500 U/g e T. reesei 240 U/g, em 5 dias de cultivo. O perfil de produção de XOs com enzimas de T. aurantiacus e T. reesei foi semelhante, o principal produto foi xilobiose, seguido por xilose, xilotriose, xilotetraose e xilopentaose, sendo esses XOs de cadeia linear. A hidrólise da hemicelulose com enzimas de A. niger produziu exclusivamente xilose, consequência da presença de elevada atividade de β-xilosidase. A velocidade de conversão da hemicelulose em XOs com as enzimas de T. reesei foi maior no início da reação (6 h), diminuindo a partir de 24 h, período em que inicia a produção de xilose. A influência da concentração de substrato e carga de xilanase na conversão da hemicelulose em XOs foi avaliada através de um planejamento experimental 22 com face centrada. A condição otimizada da hidrólise (2,6% substrato e 60 U/g de endo-β-1,4-xilanase) com o extrato de T. aurantiacus resultou em 42% de conversão em XOs. A otimização da hidrólise da hemicelulose com o extrato de T. reesei resultou em uma conversão máxima de 20%, com ótimo de 3,8 % de substrato e 87,5 U/g de endo-β-1,4-xilanase. A eficiência da hidrólise com enzimas de T. aurantiacus foi maior que a obtida com alguns extratos comerciais testados neste trabalho. Além disso, apresentaram capacidade de degradar hemiceluloses de diferentes fontes: bétula e semente de aveia, com composições variadas. Diferenças na composição de açúcares e teor de lignina não interferiram na ação dessas enzimas. A hidrólise enzimática mostrou-se mais apropriada para a produção de XOs do que a auto-hidrólise, que gerou predominantemente xilose e houve formação de furfural. Apesar do curto tempo de reação, a produção de XOs foi menor e há necessidade de purificação para obtenção de um produto final com características desejáveis.Hemicellulose extracted from sugarcane bagasse was hydrolyzed by enzymes from Thermoascus aurantiacus, Trichoderma reesei and Aspergilus niger to cause the degradation of xylan to xylooligosaccharides (XOs). Hemicellulose was extracted with hydrogen peroxide in the presence of antraquinone, sodium sulphite or hydrogen peroxide. Hemicelluloses extracted with antraquinone or sulphite presented low solubility in water, which is not appropriated to enzymatic hydrolysis. To maximize the hemicellulose yields several extraction conditions were examined applying the 24 factorial design: H2O2 concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60°C, and magnesium sulfate absence or presence (0.5%, w/v). This approach allowed selection of conditions for the extraction of low and high lignin content hemicellulose. At midpoint the yield of hemicellulose was 94.5% with more than 88% of lignin removed. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H2O2 treatment for 4 h and 20°C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. The molecular weight of hemicellulose was 21,000 g/mol with composition of aproximately 81% xylose, 4% arabinose, 4% glucose and 3% uronic acids, high water solubility (90 %). Enzymes for hemicellulose hydrolysis were produced by the fungi on wheat bran. Cellulases and hemicellulases were present in all extracts especially the endo-β-1,4-xylanase. The profile of production of XOs obtained on hydrolysis with enzymes from T. aurantiacus and T. reesei was similar, with the main product xylobiose, followed by xylose, xylotriose, xylotetraose and xylopentaose, and these XOs showed linear chain. The hydrolysis of hemicellulose with enzymes of A. niger produced exclusively xylose, a consequence of β-xylosidase content. The rate of conversion of hemicellulose in XOs with enzymes of T. reesei was higher at the beginning of the reaction (6 h), decreasing from 24 h, when starts the production of xylose. The influence of substrate concentration and loading of xylanase in conversion of hemicellulose to XOs was evaluated by an 22 full factorial design with centered face. Optimization of hydrolysis (2.6% substrate and 60 U/g endo-β-1,4-xylanase) with the extract of T. aurantiacus resulted in 42 % conversion XOs. The optimization with the extract of T. reesei resulted in a conversion of hemicellulose up to 20%, with optimal substrate 3.8% and 87.5 U/g endo-β-1,4-xylanase. The efficiency of hydrolysis by enzymes from T. aurantiacus was superior to commercial extracts, and showed ability to degrade hemicelluloses of different compositions (birchwood and oat spelt). The structural differences, such as branches and lignin content did not affect the action of these enzymes. The differences in the efficiency and extent of enzymatic hydrolysis by enzymes of these fungi might have occurred in function of differences in physicochemical properties and specific activity. The enzymatic hydrolysis was more appropriate for production of XOs than autohydrolysis, which generated predominantly xylose and formation of furfural. Despite of short reaction time, the production of XOs was low and purification is needed in order to obtain a final product with desirable characteristics

Enzymatic Cocktail Formulation for Xylan Hydrolysis into Xylose and Xylooligosaccharides

In the context of a biorefinery, lignocellulosic materials represent an important source of raw material for the bioconversion of cellulose, hemicellulose, and lignin into value-added products, such as xylose for fermentation, oligosaccharides, and bioplastics for packaging. Among the most abundant lignocellulosic materials in Brazil, sugarcane bagasse biomass stands out, as it is rich in cellulose and hemicellulose. In this context, through an experimental design, this study developed a robust enzyme cocktail containing xylanases and accessory enzymes to complete the hydrolysis of xylan from sugarcane bagasse, obtaining a low xylose yield and concentration (9% and 1.8 g/L, respectively, observed in experiment number 16 from the complete hydrolysis of a xylan assay), a fermentable sugar that is important in the production of second-generation ethanol, and a high xylooligosaccharides (XOS) yield and concentration (93.1% and 19.6 g/L, respectively, obtained from a xylooligosaccharides production assay); in general, xylan has prebiotic activities that favor an improvement in intestinal functions, with immunological and antimicrobial actions and other benefits to human health. In addition to completely hydrolyzing the sugarcane bagasse xylan, this enzymatic cocktail has great potential to be applied in other sources of lignocellulosic biomass for the conversion of xylan into xylose and XOS due to its enzymes content, involving both main chain and pendant groups hydrolysis of hemicelluloses

Xylooligosaccharides Production from Alkali-Pretreated Sugarcane Bagasse Using Xylanases from Thermoascus aurantiacus

Sugarcane bagasse hemicellulose was isolated in a one-step chemical extraction using hydrogen peroxide in alkaline media. The polysaccharide containing 80.9% xylose and small amounts of L-arabinose, 4-O-methyl-D-glucuronic acid and glucose, was hydrolyzed by crude enzymatic extracts from Thermoascus aurantiacus at 50 degrees C. Conditions of enzymatic hydrolysis leading to the best yields of xylose and xylooligosaccharides (DP 2-5) were investigated using substrate concentration in the range 0.5-3.5% (w/v), enzyme load 40-80 U/g of the substrate, and reaction time from 3 to 96 h, applying a 22 factorial design. The maximum conversion to xylooligosaccharides (37.1%) was obtained with 2.6% of substrate and xylanase load of 60 U/g. The predicted maximum yield of xylobiose by a polynomial model was 41.6%. Crude enzymatic extract of T. aurantiacus generate from sugarcane bagasse hemicellulose 39% of xylose, 59% of xylobiose, and 2% of other xylooligosaccharides.FapespCNP

Xylan Solubilization from Partially Delignified Biomass, and Residual Lignin Removal from Solubilized Xylan

Xylan is a macromolecule of industrial interest that can be solubilized from lignocellulosic materials, such as sugarcane bagasse, which is a renewable source. However, the solubilization methods of xylan need to be better developed for use in industrial applications. The main objective of this study was to evaluate xylan solubilization methods with higher yields and purity by using biomasses/fractions of sugarcane: leaf and stem, internode, node, and external fraction. Two strategies were evaluated by applying diluted sodium chlorite, sodium sulfite, and hydrogen peroxide: a delignification of the biomass before xylan solubilization; and the delignification of the solubilized xylan for residual lignin removal. The delignification of the biomass before the xylan solubilization enabled to identify material and specific conditions for yields higher than 90%. Residual lignin varied from 3.14 to 18.06%, with hydrogen peroxide in alkaline medium partial delignification shown to be effective. The delignification of xylan presented better results using diluted hydrogen peroxide, with a reduction of 58.44% of the initial lignin content. The solubilized xylans were used as a substrate for xylanase activities, resulting in higher activity than commercial xylan. In the delignification of the biomasses, hydrogen peroxide was the reagent with better results concerning the yield, purity, and solubility of the xylan. This reagent (diluted) was also better in the delignification of the solubilized xylan, resulting in lower residual lignin content. The solubility and purity tests (low salt content) indicated that the solubilized xylan presented characteristics that were similar to or even better than commercial xylan

Fruit and Restaurant Waste Polysaccharides Recycling Producing Xylooligosaccharides

A significant part of fruit production is wasted annually, a material of high value without use, causing environmental and social damage. These residues from agro-industrial processes, or those that can no longer be used in the market, can be recycled and generate value-added products by pretreatments/hydrolysis. One of the important pretreatments is acid hydrolysis, which can produce xylooligosaccharides (XOS) from biomass, a product of great commercial value in the food and pharmaceutical markets, mainly due to its prebiotic potential. Bananas, oranges, and guava generate a large volume of waste and represent much of Brazil’s fruit production. The dilute acid hydrolysis resulted in XOS production of 37.69% for banana peel, 59.60% for guava bagasse, 28.70% for orange bagasse, and 49.64% for restaurant residue. XOS were quantified by a liquid chromatograph system with a Bio-Rad Aminex HPX-87C column. The results show that, for this type of material and hydrolysis, the ideal conditions to produce XOS are high temperature, low time, and high acid concentration for banana peel residue (160 °C, 15 min, and 3% H2SO4), low temperature, low time, and high acid concentration for guava bagasse (100 °C,15 min and 3% H2SO4), high temperature and acid concentration with low time for orange bagasse (160 °C,15 min and 3% H2SO4) and high temperature and time and high acid concentration for restaurant waste (160 °C, 55 min and 3% H2SO4). This study identified acid hydrolysis conditions that maximized XOS production with a low amount of xylose production using agro-industrial and food residues, also showing the high potential of the chosen residues through the high yields of XOS production