Heterologous expression and functional characterization of a GH10 endoxylanase from \u3ci\u3eAspergillus fumigatus\u3c/i\u3e var. \u3ci\u3eniveus\u3c/i\u3e with potential biotechnological application

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-β-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60°C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMNGH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass

Heterologous expression and functional characterization of a GH10 endoxylanase from \u3ci\u3eAspergillus fumigatus\u3c/i\u3e var. \u3ci\u3eniveus\u3c/i\u3e with potential biotechnological application

Xylanases decrease the xylan content in pretreated biomass releasing it from hemicellulose, thus improving the accessibility of cellulose for cellulases. In this work, an endo-β-1,4-xylanase from Aspergillus fumigatus var. niveus (AFUMN-GH10) was successfully expressed. The structural analysis and biochemical characterization showed this AFUMN-GH10 does not contain a carbohydrate-binding module. The enzyme retained its activity in a pH range from 4.5 to 7.0, with an optimal temperature at 60°C. AFUMN-GH10 showed the highest activity in beechwood xylan. The mode of action of AFUMNGH10 was investigated by hydrolysis of APTS-labeled xylohexaose, which resulted in xylotriose and xylobiose as the main products. AFUMN-GH10 released 27% of residual xylan from hydrothermally-pretreated corn stover and 14% of residual xylan from hydrothermally-pretreated sugarcane bagasse. The results showed that environmentally friendly pretreatment followed by enzymatic hydrolysis with AFUMN-GH10 in low concentration is a suitable method to remove part of residual and recalcitrant hemicellulose from biomass

Produção de xilanases por Penicillium janthinellum E aplicação das enzimas no branqueamento de polpas kraft

Orientador: Nora Marcela Haun QuirosTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Neste estudo investigou ¿ se a possibilidade de utilização de um hidrolisado hemicelulósico, obtido a partir da hidrólise ácida branda do bagaço de cana-de-açúcar, como meio de cultura para crescimento de fungo Penicillium janthinellum e para produção de xilanases. As condições ótimas para crescimento do fungo e produção de xilanases em incubadoras com movimento rotatório foram: temperatura de 30ºC, pH inicial entre 5,0 e 6,0 e agitação 60 rpm.Sob essas condições, o crescimento celular esteve associado à síntese das enzimas b-xilosidades, b-glaucosidades, endoglucanases e exoglucanases foram pequenos. A produção de xilanases foi também realizada em fermentador de 4 l agitado mecanicamente em meio à base de hidrolisado hemicelulósico, tendo a atividade das xilanases assim produzidas se mostrado menor que a das obtidas em incubadora com movimento rotatório. Uma endoxilanase e uma b-xilosidase foram purificadas do fluido de cultivo do fungo. O estudo da influência do pH revelou um pH ótimo de 5,5 para a atividade da endoxilanase e de 6,0 para a b-xilosidase. Os valores ótimos de temperatura foram 50ºC e 60ºC para endoxilanase e b-xilosidase, respectivamente. Os principais produtos da hidrólise de xilana pela endoxilanase foram xilose, xilobiose e xilotriose. Os pesos moleculares da endoxilanas e b-xilosidade, determinados pó SDS-PAGE, foram 54 kDa e 74,3 kDa, respectivamente... Observação: O resumo, na íntegra, poderá ser visualizado no texto completo da tese digitalAbstract: This work investigates the possibility of utilizing a hemicellullosic hydrolysate obtained from mild acid hydrolysis of sugar cane bagasse as a culture medium for growing the fungus Penicillium janthinellum and for producing xylanases. The optimum conditions for the growth of fungus and the production of xylanases in a rotary incubator were: temperature of 30ºC, initial pH between 5.0 and 6.0 and agitation at 60 rpm. Under these conditions, cell growth was associated with the induction levels of the enzymes b-xylosidadses, b-glucosidases, endoglucanases and exoglucanases werw small. Xylanase production was also carried out in a 4 L mechanically agitated fermentor, using a hemicellulosic hidrolysate-based medium. The activity of the xylanases produced in this manner was lower than that of the xylanases pçoduced in the rotary incubator. An endoxylanase and a b-xylosidase were purified from the culture filtrate of the fungus. The study of the pH influence revealed an optimum pH 5.5 for endoxylanase activity and 6.0 for b-xylosidase. The optimum values of temperature were 50ºC and 60ºC for endoxylanase and b-xylosidase, respectively. The main products of xylan hydrolysis by endoxylanase were xylose, xylobiose and xylotriose... Note: The complete abstract is available with the full electronic digital thesis or dissertationsDoutoradoBioquimicaDoutor em Ciências Biológica

<b>Uso de carvão ativado e resina de troca iônica para limpeza e concentração de enzimas em extratos de madeira biodegradada</b> - 10.4025/actascitechnol.v32i4.5671

<em>Ceriporiopsis subvermispora</em> foi utilizado para a biodegradação de cavacos de <em>Eucalyptus grandis</em> na presença ou ausência de cossubstratos (glicose e milhocina) durante 7, 14 e 28 dias. Os cavacos obtidos em cada um desses períodos foram extraídos com tampão acetato de sódio 50 mM (pH 5,5) adicionado de 0,01% de Tween 60. A atividade de manganês peroxidases (MnPs) nos extratos foi marcante ao longo de todo o período de biodegradação, tanto na ausência (430, 765 e 896 UI kg^-1, respectivamente) quanto na presença de cossubstratos (1.013, 2.066 e 2.323 UI kg^-1, respectivamente). Os extratos apresentaram elevadas relações entre absorbância a 280 e a 405 nm, indicando forte abundância de compostos aromáticos derivados da lignina em relação à heme-peroxidases. A adsorção em carvão ativado se mostrou uma estratégia adequada para reduzir a absorbância a 280 nm em todos os extratos. Além disso, permitiu maximizar a capacidade de um leito de resina trocadora de ânions (DEAE Sepharose), utilizado para concentrar as MnPs presentes nos referidos extratos. Concluiu-se que o uso de carvão ativado seguido de adsorção em DEAE Sepharose é uma estratégia que pode ser utilizada para concentrar MnPs em extratos obtidos durante a biodegradação de <em>E. grandis</em> por <em>C. subvermispora</em>

Search for optimum conditions of sugarcane bagasse hemicellulose extraction

A process has been elaborated for one-step low lignin content sugarcane bagasse hemicellulose extraction using alkaline solution of hydrogen peroxide. To maximize the hemicellulose yields several extraction conditions were examined applying the 2(4) factorial design: H(2)O(2) concentration from 2 to 6% (w/v), reaction time from 4 to 16 h, temperature from 20 to 60 degrees 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. Lignin removal is suppressed at low extraction temperatures and in the absence of magnesium sulfate. Hemicellulose in 86% yield with low lignin content (5.9%) was obtained with 6% H(2)O(2) treatment for 4 h and 20 degrees C. This hemicellulose is much lighter in color than samples obtained at the midpoint condition and was found suitable for subsequent enzymatic hydrolysis. (C) 2009 Elsevier B.V. All rights reserved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo-FAPESP[06/03564-9]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo-FAPESP[08/03204-8

Induction of cellulase and hemicellulase activities of Thermoascus aurantiacus by xylan hydrolyzed products

Thermoascus aurantiacus is able to secrete most of the hemicellulolytic and cellulolytic enzymes. To establish the xylanase inducers of T. aurantiacus, the mycelia were first grown on glucose up until the end of the exponential growth phase, followed by washing and re-suspension in a basal medium without a carbon source. Pre-weighed amounts of xylose (final concentration of 3.5 mg/ml), xylobiose (7 mg/ml) and hydrolyzed xylan from sugarcane bagasse (HXSB) which contained xylose, xylobiose and xylotriose (6.8 mg/ml) were evaluated as inducers of xylanase. It was observed that xylose did not suppress enzyme induction of T. aurantiacus when used in low concentrations, regardless of whether it was inoculated with xylobiose. Xylobiose promoted fast enzyme production stopping after 10 h, even at a low consumption rate of the carbon source; therefore xylobiose appears to be the natural inducer of xylanase. In HXSB only a negligible xylanase activity was determined. Xylose present in HXSB was consumed within the first 10 h while xylobiose was partially hydrolyzed at a slow rate. The profile of alpha-arabinofuranosidase induction was very similar in media induced with xylobiose or HXSB, but induction with xylose showed some positive effects as well. The production profile for the xylanase was accompanied by low levels of cellulolytic activity. In comparison, growth in HXSB resulted in different profiles of both xylanase and cellulase production, excluding the possibility of xylanase acting as endoglucanases.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP) [06/035649]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Conselho Nacional Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional Desenvolvimento Cientifico e Tecnologico (CNPq

Uma visão sobre a estrutura, composição e biodegradação da madeira A vision of wood structure, composition and biodegradation

<abstract language="eng">Wood is the main raw material used in the pulp and paper industry. It is a material that presents heterogeneous structure and complex composition, which results in a relatively resistant material to the biodegradation process. In the present review, we attempted to summarize the structural characteristics of wood and describe the chemical nature of its major components to, afterwards, comment about its biodegradation. The role of the enzyme manganese peroxidase in the lignin degradation by a selective white-rot fungus, Ceriporiopsis subvermispora, was highlighted

Enzymatic Hydrolysis of Chemithermomechanically Pretreated Sugarcane Bagasse and Samples with Reduced Initial Lignin Content

Chemithermomechanical (CTM) processing was used to pretreat sugarcane bagasse with the aim of increasing cell wall accessibility to hydrolytic enzymes. Yields of the pretreated samples were in the range of 75-94%. Disk refining and alkaline-CTM and alkaline/sulfite-CTM pretreatments yielded pretreated materials with 21.7, 17.8, and 15.3% of lignin, respectively. Hemicellulose content was also decreased to some extent. Fibers of the pretreated materials presented some external fibrillation, fiber curling, increased swelling, and high water retention capacity. Cellulose conversion of the alkaline-CTM- and alkaline/sulfite-CTM-pretreated samples reached 50 and 85%, respectively, after 96 h of enzymatic hydrolysis. Two samples with low initial lignin content were also evaluated after the mildest alkaline-CTM pretreatment. One sample was a partially delignified mill-processed bagasse. The other was a sugarcane hybrid selected in a breeding program. Samples with lower initial lignin content were hydrolyzed considerably faster in the first 24 h of enzymatic digestion. For example, enzymatic hydrolysis of the sample with the lowest initial lignin content (14.2%) reached 64% cellulose conversion after only 24 h of hydrolysis when compared with the 30% observed for the mill-processed bagasse containing an initial lignin content of 24.4%. (C) 2011 American Institute of Chemical Engineers Biotechnol. Prog., 27: 395-401, 2011FAPESP[08/56256-5]CNPqCAPE

Characterization of commercial cellulases and their use in the saccharification of a sugarcane bagasse sample pretreated with dilute sulfuric acid

This study aimed to correlate the efficiency of enzymatic hydrolysis of the cellulose contained in a sugarcane bagasse sample pretreated with dilute H(2)SO(4) with the levels of independent variables such as initial content of solids and loadings of enzymes and surfactant (Tween 20), for two cellulolytic commercial preparations. The preparations, designated cellulase I and cellulase II, were characterized regarding the activities of total cellulases, endoglucanase, cellobiohydrolase, cellobiase, beta-glucosidase, xylanase, and phenoloxidases (laccase, manganese and lignin peroxidases), as well as protein contents. Both extracts showed complete cellulolytic complexes and considerable activities of xylanases, without activities of phenoloxidases. For the enzymatic hydrolyses, two 2(3) central composite full factorial designs were employed to evaluate the effects caused by the initial content of solids (1.19-4.81%, w/w) and loadings of enzymes (1.9-38.1 FPU/g bagasse) and Tween 20 (0.0-0.1 g/g bagasse) on the cellulose digestibility. Within 24 h of enzymatic hydrolysis, all three independent variables influenced the conversion of cellulose by cellulase I. Using cellulase II, only enzyme and surfactant loadings showed significant effects on cellulose conversion. An additional experiment demonstrated the possibility of increasing the initial content of solids to values much higher than 4.81% (w/w) without compromising the efficiency of cellulose conversion, consequently improving the glucose concentration in the hydrolysate.FAPESPCNPqCAPE