RESUMO
Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.
Assuntos
Azacitidina/análogos & derivados , Repressão Catabólica/efeitos dos fármacos , Celulase/biossíntese , Inibidores Enzimáticos/metabolismo , Enzimas/metabolismo , Sordariales/efeitos dos fármacos , Xilosidases/biossíntese , Azacitidina/metabolismo , Decitabina , Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Sordariales/crescimento & desenvolvimento , Triticum/metabolismo , Triticum/microbiologiaRESUMO
One full-length ß-xylosidase gene (hxylA) was identified from the Humicola grisea var. thermoidea genome and the cDNA was successfully expressed by Pichia pastoris SMD1168. An optimization of enzyme production was carried out, and methanol was found to be the most important parameter. The purified enzyme was characterized and showed the optimal conditions for the highest activity at pH 7.0 and 50°C, being thermostable by maintaining 41% of its activity after 12h incubated at 50°C. HXYLA is a bifunctional enzyme; it showed both ß-xylosidase and α-arabinfuranosidase activities. The Km and Vmax values were 1.3mM and 39.1U/mg, respectively, against 4-nitrophenyl ß-xylopyranoside. HXYLA showed a relatively strong tolerance to xylose with high Ki value of 603mM, with the xylose being a non-competitive inhibitor. HXYLA was successfully used simultaneously and sequentially with an endo-xylanase for analysis of synergism in the degradation of commercial xylans. Furthermore, commercial cellulases supplementation with HXYLA during sugarcane bagasse hydrolysis increased hydrolysis in 29%. HXYLA is distinguished from other ß-xylosidases by the attractive characteristics for industrial applications such as thermostability, high tolerance xylose and saccharification of biomass by convert xylan into fementable monosaccharides and improve cellulose hydrolysis.
Assuntos
Celulose/metabolismo , Proteínas Recombinantes/metabolismo , Saccharum/química , Xilose/farmacologia , Xilosidases/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Metais/farmacologia , Pichia/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Análise de Sequência , Especificidade por Substrato , Xilosidases/química , Xilosidases/genéticaRESUMO
Humicola grisea var. thermoidea produces two forms of extracellular xylanase. The component form 1 was purified using the electroelution method, due to the very small production of this extracellular enzyme. The apparent molecular mass was 61.8 kDa by SDS-PAGE.
Humicola grisea var. thermoidea produz duas formas de xilanase extracelular. A componente forma 1 foi purificada usando o método de eletroeluição devido à baixa produção desta enzima extracelular. A aparente massa molar foi determinada 61,8 kDa por SDS-PAGE.