Caracterização bioquímica da endoxilanase recombinante (HXYN2r) do fungo termofílico Humicola grisea var. thermoidea e sua aplicação na sacarificação de resíduos agrícolas
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2008-06-30
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Universidade Federal de Goiás
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Xylanases have been used in the biobleaching of paper pulps, in bioconversion of
plant biomass, for food and feed industries, among others. For successful selection
of xylanases suitable for specific industrial applications it is important to
characterize enzymes isolated from different sources. The thermophilic fungus
Humicola grisea var. thermoidea is described as a good producer of extracellular
endoxylanases and the Hxyn2 gene from this fungus was isolated and expressed in
yeast Pichia pastoris. The aim of this project was focused on the production,
purification and biochemical characterization of the recombinant HXYN2 (HXYN2r)
enzyme and application on enzymatic hydrolysis of lignocellulosics substrates.
The culture conditions of P. pastoris in flasks, using the 12.3 transformant
and BMMY-U medium, were optimized and the best xylanolytic activity was
478.2 U/mL after 96 h, with a protein concentration of 100 mg/L, using
2.34% (w/v) of nitrogen sources (yeast extract plus urea 1.34:1.0% - w/v),
1% (v/v) of methanol, and OD600 of 10. The HXYN2r enzyme was purified by gel
filtration chromatography with a yield of 6.4% and showed optimum pH and
temperature values of 6.5 and 60ºC, respectively, maintaining 100% of initial
activity after 4 h of incubation at pH 5.5, 6.5 and 7.5. The half-life time was 18 min
at 60ºC and the enzyme was 100% stable after 3 h of incubation at 50ºC. Km and
Vmax values were 7.9 mg/mL e 235.4 μmol/(mL.min), respectively. The HXYN2r
enzyme were used on the enzymatic hydrolysis of sugarcane bagasse (SCB), corn cob (CC) and foliar sample of sugarcane (FSC) alone or with the enzymes of
xylanolytic and cellulolytic system produced by H. grisea fungus. For enzymatic
hydrolysis experiments, the substrates were milled and pretreated with
0.25% (w/v) of H2SO4 by 30 min. On the enzymatic hydrolysis the best conversion
yield of hemicellulosic fractions were obtained using PpHXYN2r supplemented
with EHg: 42.8% to CC, 9.6% to SCB and, a mean of 20% to foliar samples.
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CARVALHO, Wagner Rodrigues de. Caracterização bioquímica da endoxilanase recombinante (HXYN2r) do fungo termofílico Humicola grisea var. thermoidea e sua aplicação na sacarificação de resíduos agrícolas. 2008. 143 f. Tese (Doutorado em Ciências da Saúde) - Universidade Federal de Goiás, Goiânia, 2008.