Análise do perfil de adsorção das enzimas envolvidas na sacarificação enzimática de diferentes bagaços de cana-de-açúcar pré-tratados com sulfito ácido

A produção de etanol de segunda geração é uma realidade brasileira que já vem sendo implementada na indústria. Porém, vários fatores que governam a sacarificação enzimática de materiais lignocelulósicos ainda necessitam ser melhor esclarecidos, de forma a baixar o custo e aumentar o rendimento final de monossacarídeos. A adsorção das enzimas durante a sacarificação da celulose é um fator determinante para uma hidrólise eficiente, que pode ser influenciada pelas características do substrato lignocelulósico, como conteúdo ou distribuição de hemicelulose e lignina. Diante disso, este trabalho teve como objetivo avaliar o perfil de adsorção das enzimas envolvidas na sacarificação do bagaço de cana-de-açúcar pré-tratado com sulfito ácido em diferentes temperaturas. Os resultados revelaram que a adsorção varia com as características finais dos substratos gerados, como o teor de lignina remanescente e a quantidade de grupos sulfônicos

Laccase production by free and immobilized fungal mycelium of Trametes versicolor

The production of laccase by free and immobilized mycelium of Trametes versicolor was evaluated. Fermentation experiments were carried out using a Trametes defined medium [1] supplemented with tween-80 (0.5%, w/v) and xylidine (30 μM) to stimulate and induce the secretion of extracellular enzyme. The support for the mycelium immobilization consisted in 0.3 cm3 cubes of synthetic fiber (Scotch Brite, 3M Spain, SA), which were added to the culture system at 1 g/100 mL of medium. Assays were performed in 250-mL Erlenmeyer flasks and in a 2 L stirred tank bioreactor. For the flasks fermentations, three 7 mm diameter plugs from the fungal monoculture plate were inoculated into 50 mL culture medium in the presence or not of the immobilization support. For the assays in bioreactor, the volume of inoculum necessary to obtain an initial cell concentration of 70 mg/L was transferred to the reactor containing 1 L of culture medium with or without the immobilization support. In both cases, the fungus was incubated at 28 °C and 180 rpm. During the experiments, samples were periodically withdrawn for laccase and glucose determinations. Synthetic fiber was used as immobilization support since this material was demonstrated to be of great potential for fungi immobilization [2]. Additionally, many studies have demonstrated that fermentation systems with immobilized cells are able to increase the process productivity. However, the laccase production by T. versicolor (present study) did not show this performance. In both systems (Erlenmeyer flasks and bioreactor), the highest laccase production was obtained when using free mycelium. Additionally, the maximum laccase production obtained in bioreactor was lower than the maximum found in Erlenmeyer-flasks, suggesting that the conditions used in the bioreactor should be optimized to increase the laccase production results. Due to the great importance of the laccases in the industrial sector, more studies will be performed aiming to find a strategy to maximize the production of this enzyme by T. versicolo

Laccase production by free and immobilized mycelia of Peniophora cinerea and Trametes versicolor: a comparative study

The production of laccase by immobilized mycelia of Peniophora cinerea and Trametes versicolor was studied. In an initial stage, experimental assays were performed in Erlenmeyer flasks using free and immobilized mycelium, and the performance of the fungal strains to produce the enzyme was compared. Both fungi adhered into the support material (a synthetic fiber), growing not only on the surface but also in the interspaces of the fibers. Immobilization of P. cinerea provided a 35-fold increase in laccase production when compared to the production obtained by using free mycelium. On the other hand, immobilization of T. versicolor caused a decrease in laccase activity. A comparison between the strains revealed that immobilized P. cinerea (3,500 U/L) surpassed the enzyme production by free T. versicolor (800 U/L). When the conditions that gave the best laccase production to each fungus were employed in a stirred tank bioreactor, very low laccase production was observed for both the cases, suggesting that shear stress and mycelia damage caused by the agitation impellers negatively affected the enzyme production.S.C. Silverio acknowledges the financial support from FCT (Fundacao para a Ciencia e para a Tecnologia, Ph.D. grant SFRH/BD/43439/2008), Portugal; and S. Moreira acknowledges CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnologico), Brazil

Effects of enzymatic removal of plant cell wall acylation (acetylation, p-coumaroylation, and feruloylation) on accessibility of cellulose and xylan in natural (non-pretreated) sugar cane fractions

Background: Sugar cane internodes can be divided diagonally into four fractions, of which the two innermost ones are the least recalcitrant pith and the moderately accessible pith-rind interface. These fractions differ in enzymatic hydrolyzability due to structural differences. In general, cellulose hydrolysis in plants is hindered by its physical interaction with hemicellulose and lignin. Lignin is believed to be linked covalently to hemicellulose through hydroxycinnamic acids, forming a compact matrix around the polysaccharides. Acetyl xylan esterase and three feruloyl esterases were evaluated for their potential to fragment the lignocellulosic network in sugar cane and to indirectly increase the accessibility of cellulose. Results: The hydrolyzability of the pith and pith-rind interface fractions of a low-lignin-containing sugar cane clone (H58) was compared to that of a reference cultivar (RC). Acetyl xylan esterase enhanced the rate and overall yield of cellulose and xylan hydrolysis in all four substrates. Of the three feruloyl esterases tested, only TsFaeC was capable of releasing p-coumaric acid, while AnFaeA and NcFaeD released ferulic acid from both the pith and interface fractions. Ferulic acid release was higher from the less recalcitrant clone (H58)/fraction (pith), whereas more p-coumaric acid was released from the clone (RC)/fraction (interface) with a higher lignin content. In addition, a compositional analysis of the four fractions revealed that p-coumaroyl content correlated with lignin, while feruloyl content correlated with arabinose content, suggesting different esterification patterns of these two hydroxycinnamic acids. Despite the extensive release of phenolic acids, feruloyl esterases only moderately promoted enzyme access to cellulose or xylan. Conclusions: Acetyl xylan esterase TrAXE was more efficient in enhancing the overall saccharification of sugar cane, compared to the feruloyl esterases AnFaeA, TsFaeC, and NcFaeD. The hydroxycinnamic acid composition of sugar cane fractions and the hydrolysis data together suggest that feruloyl groups are more likely to decorate xylan, while p-coumaroyl groups are rather linked to lignin. The three different feruloyl esterases had distinct product profiles on non-pretreated sugar cane substrate, indicating that sugar cane pith could function as a possible natural substrate for feruloyl esterase activity measurements. Hydrolysis data suggest that TsFaeC was able to release p-coumaroyl groups esterifying lignin.Peer reviewe

Exploring glycoside hydrolases and accessory proteins from wood decay fungi to enhance sugarcane bagasse saccharification

Abstract\ud \ud Background\ud Glycoside hydrolases (GHs) and accessory proteins are key components for efficient and cost-effective enzymatic hydrolysis of polysaccharides in modern, biochemically based biorefineries. Currently, commercialized GHs and accessory proteins are produced by ascomycetes. However, the role of wood decay basidiomycetes proteins in biomass saccharification has not been extensively pursued. Wood decay fungi degrade polysaccharides in highly lignified tissues in natural environments, and are a promising enzyme source for improving enzymatic cocktails that are designed for in vitro lignocellulose conversion.\ud \ud \ud Results\ud GHs and accessory proteins were produced by representative brown- and white-rot fungi, Laetiporus sulphureus and Pleurotus ostreatus, respectively. Concentrated protein extracts were then used to amend commercial enzymatic cocktails for saccharification of alkaline-sulfite pretreated sugarcane bagasse. The main enzymatic activities found in the wood decay fungal protein extracts were attributed to endoglucanases, xylanases and β-glucosidases. Cellobiohydrolase (CBH) activities in the L. sulphureus and P. ostreatus extracts were low and nonexistent, respectively. The initial glucan conversion rates were boosted when the wood decay fungal proteins were used to replace half of the enzymes from the commercial cocktails. L. sulphureus proteins increased the glucan conversion levels, with values above those observed for the full load of commercial enzymes. Wood decay fungal proteins also enhanced the xylan conversion efficiency due to their high xylanase activities. Proteomic studies revealed 104 and 45 different proteins in the P. ostreatus and L. sulphureus extracts, respectively. The enhancement of the saccharification of alkaline-pretreated substrates by the modified enzymatic cocktails was attributed to the following protein families: GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.\ud \ud \ud Conclusions\ud The extracellular proteins produced by wood decay fungi provide useful tools to improve commercial enzyme cocktails that are currently used for the saccharification of alkaline-pretreated lignocellulosic substrates. The relevant proteins encompass multiple glycoside hydrolase families, including the GH5- and GH45-endoglucanases, GH3-β-glucosidases, and GH10-xylanases.The authors thank J.M. Silva and J.C. Tavares for technical assistance. This work\ud was supported by FAPESP (contract numbers 08/56256-5 and 2014/06923-6),\ud CNPq (contract numbers 442333/2014-5; 310186/2014-5 and 140796/2013-\ud 4), and CAPES. We gratefully acknowledge the provision of time on the MAS\ud and NGS facilities (LNBio and CTBE, respectively) at the National Center for\ud Research in Energy and Materials (CNPEM).\ud The work was supported by Fundação de Amparo à Pesquisa do Estado de\ud São Paulo (FAPESP), contract numbers 08/56256-5 and 2014/06923-6, and by\ud Conselho Nacional de Pesquisa (CNPq), contract numbers 442333/2014-5;\ud 310186/2014-5, 140796/2013-4

The effect of agitation speed, enzyme loading and substrate concentration on enzymatic hydrolysis of cellulose from brewer’s spent grain

Brewer’s spent grain components (cellulose, hemicellulose and lignin) were fractionated in a two-step chemical pretreatment process using dilute sulfuric acid and sodium hydroxide solutions. The cellulose pulp produced was hydrolyzed with a cellulolytic complex, Celluclast 1.5 L, at 45 ºC to convert the cellulose into glucose. Several conditions were examined: agitation speed (100, 150 and 200 rpm), enzyme loading (5, 25 and 45 FPU/g substrate), and substrate concentration (2, 5 and 8% w/v), according to a 2 3 full factorial design aiming to maximize the glucose yield. The obtained results were interpreted by analysis of variance and response surface methodology. The optimal conditions for enzymatic hydrolysis of brewer’s spent grain were identified as 100 rpm, 45 FPU/g and 2% w/v substrate. Under these conditions, a glucose yield of 93.1% and a cellulose conversion (into glucose and cellobiose) of 99.4% was achieved. The easiness of glucose release from BSG makes this substrate a raw material with great potential to be used in bioconversion processes.Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo), Brazil. Novozymes ( FAPESP )Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq