The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.

Characterization of a recombinant xylose tolerant ß-xylosidase from Humicola grisea var. thermoidea and its use in sugarcane bagasse hydrolysis.

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.

Transcriptional profile of the human pathogenic fungus Paracoccidioides lutzii in response to sulfamethoxazole.

Paracoccidioidomycosis (PCM) is the most prevalent mycosis in Latin America and is caused by a group of fungi within the Paracoccidioides genus. The disease may present clinical and pathological manifestations ranging from asymptomatic pneumonia pulmonary lesions, to disseminated forms involving multiple organs. Sulfonamides were the first drugs used to treat PCM and are still used against this fungal infection. Sulfa drugs are competitive antagonists of ρ-aminobenzoic acid (PABA), a reaction catalyzed by dihydropteroate synthase (DHPS). However, the molecular effects of sulfonamides against the Paracoccidioides genus are unknown. The aim of this work was to investigate the global mechanism of action of sulfamethoxazole on Paracoccidioides lutzii. Yeast cells were grown on minimum medium in the presence or absence of sulfamethoxazole to construct EST libraries. The representational difference analysis (RDA) technique was used to identify up- and down-regulated P. lutzii genes after treatment with sulfamethoxazole. Approximately six transcripts related to mitochondrial function were differentially expressed. To confirm the RDA and bioinformatics results, several relevant genes were studied with quantitative real-time polymerase chain reaction (qRT-PCR) to evaluate their levels of expression. To confirm the impact of sulfamethoxazole on mitochondria, we measured the reduction of tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) by P. lutzii with or without exposure to the drug. MTT assays reveal that sulfamethoxazole produces a marked dose-dependent adverse effect on P. lutzii. The transcriptional activity of selected genes in infected macrophages corroborated our in vitro results. The results indicated that sulfamethoxazole acts in P. lutzii as a competitor for amino acid, nucleic acids and folate cofactor biosynthesis, disrupting mitochondrial functions.