Produção de xilanases por fermentação em estado sólido de farelo de trigo utilizando consórcios fúngicos / Production of xylanases by solid state fermentation of wheat bran using fungal consortia

A fim de se agregar valor ao resíduo farelo de trigo gerado em indústrias do setor alimentício avaliou-se, no presente trabalho, o potencial deste subproduto como substrato para produção de enzima xilanase no cultivo em estado sólido, utilizando consórcios fúngicos bem como os fungos Aspergillus oryzae CCT nº 0975 (ATCC9362) eTrichoderma reesei CCT nº 2768 - QM 9414. Para tanto utilizou-se o farelo de trigo, não lavado e não autoclavado, como fonte de carbono e energia na fermentação em estado sólido pelo fungo Aspergillus oryzae que apresentou maior produção do percentual de proteína nas 72 horas de cultivo. Depois de realizado um Delineamento Composto Central Rotacional (DCCR) - planejamento fatorial 23,com três repetições no ponto central e seis pontos axiais - partiu-se para otimização dos fatores que foram considerados significativos no processo: umidade, pH e granulometria. Os fatores foram considerados significativos pela A NOVA com o nível de 95% de confiança e com o resultado otimizado de atividade enzimática de (1.84 ± 0.01) UI/mL utilizando pH 3,3, granulometria de 900,0 µm e umidade de 40%. O caldo enzimático obtido foi considerado eficiente na modificação de tipificação de farinhas de trigo pelo estudo dos parâmetros reológicos do falling number e alveografia sendo estável por cerca de 3 meses

This study aimed to find alternatives for wheat bran disposal destination generated in food industry sector,thus contributing to the reduction of the resultant impact of residue depo-sition in the environment. The poten-tial of the wheat bran as a substrate for xylanase production by solid-state fermentation using fungal con-sortiums as well as Aspergillus ory-zae (ATCC9362) and Trichoderma reesei (2768) was valued. The use of non-washed and non-autoclaved wheat bran as carbon and energy source in solid-state fermentation by A. oryzae fungus showed greater per-centage of produced protein after 72 h of cultivation. The use of a central composite rotatable design(CCRD), 23 factorial planning with three rep-etitions at the central point as well as six axial points, coupled with Sur-face Response Methodology (SRM) allowed to assay the influence of hu-midity, pH, and grain size (indepen-dent variables or factors) on the xy-lanase activity(dependent variable or response) as well as to optimize the best conditions for the enzyme production. The results showed that all factors and their combinations were significant at 95% confidence level. The optimized xylanase activi-ty was (1.84 ± 0.01) UI/mL, obtained at 40% humidity and pH 3.3 with a grain size of 900.0 µm. The produced broth was stable for 3 months and approximately had 50% of the initial xylanase activity at 4°C. SDS-PAGE assay showed that xylanase has 30 kDa molar mass. The obtained en-zymatic broth was efficient to modify wheat flours as shown by the falling number rheologic parameters and alveography assay

Improving the thermostability of Trichoderma reesei xylanase 2 by introducing disulfide bonds

Background: Xylanases are considered one of the most important enzymes in many industries. However, their low thermostability hampers their applications in feed pelleting, pulp bleaching, and so on. The main aim of this work was to improve the thermostability of Trichoderma ressei xylanase 2 (Xyn2) by introducing disulfide bonds between the N-terminal and α-helix and the ß-sheet core. Results: In this work, two disulfide bonds were separately introduced in the Xyn2 to connect the N-terminal and α-helix to the ß-sheet core of Xyn2. The two disulfide bonds were introduced by site-directed mutagenesis of the corresponding residues. The half-life of the mutants Xyn2C14­52 (disulfide bond between ß-sheets B2 and B3) and Xyn2C59­149 (disulfide bond between ß-sheets A5 and A6) at 60°C was improved by approximately 2.5- and 1.8-fold compared to that of the wild type Xyn2. In addition, the enzyme's resistance to alkali and acid was enhanced. Conclusion: Our results indicated that the connection of the N-terminal and α-helix to the ß-sheet core is due to the stable structure of the entire protein.

Xylanolytic enzymes production by Aspergillus niger GS1 from solid-state fermentation on corn stover and their effect on ruminal digestibility

Hemicellulosic agricultural by-products such as corn stover (CS) are highly available materials which represent an opportunity to develop value added products. Native Aspergillus niger GS1 was used for solid-state fermentation (SSF) on alkali pre-treated CS (ACS) aimed to optimize xylanolytic enzymes production, and their effect on in vitro ruminal and true digestibility of ACS. Enzyme production was empirically modelled using a fractional factorial design 2(9-5), and the resulting significant factors were glucose, yeast extract and two mineral salts, which were arranged in a Draper-Lin optimization design at two levels. Predicted optimum xylanolytic activity of 33.6 U (mg protein)-1 was achieved at 48 hrs of SSF, and was validated by confirmatory experiments. ACS was incubated with a semipurified enzymatic extract (EE) showing a xylanolytic activity of 1600 U kg-1 dry ACS for 12 hrs before exposure to cow's ruminal liquid for 72 hrs, which led to 5 percent and 10 percent increase of in vitro ruminal and true digestibility, respectively. CS is a readily available by-product in different regions which after alkaline treatment and partial hydrolysis with the EE, may be advantageously used as supplement for ruminant feed.

Hydrolysis of xylans by enzyme systems from solid cultures of Trichoderma harzianum strains

Xylanase activity was isolated from crude extracts of Trichoderma harzianum strains C and 4 grown at 28oC in a solid medium containing wheat bran as the carbon source. Enzyme activity was demonstrable in the permeate after ultrafiltration of the crude extracts using an Amicon system. The hydrolysis patterns of different xylans and paper pulps by xylanase activity ranged from xylose, xylobiose and xylotriose to higher xylooligosaccharides. A purified ß-xylosidase from the Trichoderma harzianum strain released xylose, xylobiose and xylotriose from seaweed, deacetylated, oat spelt and birchwood xylans. The purified enzyme was not active against acetylated xylan and catalyzed the hydrolysis of xylooligosaccharides, including xylotriose, xylotetraose and xylopentaose. However, the enzyme was not able to degrade xylohexaose. Xylanase pretreatment was effective for hardwood kraft pulp bleaching. Hardwood kraft pulp bleached in the XEOP sequence had its kappa number reduced from 13.2 to 8.9 and a viscosity of 20.45 cp. The efficiency of delignification was 33