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Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, Programa de Pós-Graduação em Biologia Molecular, 2016. A lignocelulose é o principal componente da biomassa vegetal, sendo constituída por celulose, hemicelulose e lignina. A xilana é o principal componente da hemicelulose e para a sua completa degradação é necessário a ação cooperativa de um sistema composto por várias enzimas, incluindo endo-xilanases (XYN), β-xilosidases (XYL) e α-L-arabinofuranosidases (ABF). No presente trabalho, foi realizada a produção de hemicelulases recombinantes e a aplicação na hidrólise do bagaço de cana-de-açúcar (BCA) pré-tratado por explosão a vapor. As primeiras enzimas produzidas foram duas XYLs do fungo Humicola grisea var. thermoidea, sendo esse o primeiro trabalho a reportar a caracterização de genes de XYLs de H. grisea, bem como, a sua expressão heteróloga e caracterização bioquímica. Os dois genes, hxylA e hxylB, foram identificados a partir do genoma de H. grisea, um com 984 pb e outro com 1617 pb, respectivamente. Nenhum íntron e peptídio sinal foi encontrado e o domínio catalítico de GH43 estava presente em ambas. Os cDNAs correspondente as duas enzimas foram utilizados para a transformar duas linhagens de Pichia pastoris, GS115 e SMD1168. Uma otimização da produção da enzima denominada HXYLA foi realizada por meio de planejamento experimental 23. A influência da densidade celular inicial, concentração de metanol e fonte de nitrogênio foi avaliada e o parâmetro mais significativo para a produção foi a concentração de metanol. A enzima HXYLA purificada foi utilizada para a realização da caracterização enzimática quanto ao pH e temperatura ótimos (7,0 e 50 °C), efeito de íons e reagentes químicos na atividade de HXYLA, substrato específico e parâmetros cinéticos. A enzima HXYLA apresentou atividade de β- xilosidase e α-L-arabinofuranosidase, sendo uma enzima bifuncional e altamente tolerante a xilose, com elevado valor de Ki (350 mM). Além das β-xilosidases, foi produzida e purificada uma endoxilanase (HXYN2) recombinante de H. grisea a partir de um clone de P. pastoris obtido pelo grupo de pesquisa do laboratório de Biotecnologia de Fungos (LBF/UFG). Além dessas hemicelulases, uma α- L-arabinofuranosidase (ABF3) de Penicillium purpurogenum teve seu cDNA introduzido em P. pastoris e a enzima foi produzida com sucesso. Após a produção hetoróloga das enzimas, foi utilizado um planejamento experimental 23 para avaliar a melhor formulação contendo as três hemicelulases recombinantes (HXYN2, ABF3 e HXYLA) para a hidrólise do BCA pré-tratado por explosão a vapor, sendo possível estabelecer uma mistura capaz de realizar a conversão da fração residual de xilana em xilose, sendo a bioconversão eficiente da fração de xilana do BCA uma etapa imprescindível para o aproveitamento da xilose. A melhor formulação contendo as três hemicelulases recombinantes foi obtido com a menor concentração de ABF3, sendo que essa enzima foi a que exerceu efeito mais significativo durante a hidrólise do BCA. Além disso, o coquetel contendo as hemicelulases recombinantes foi utilizado para suplementar uma mistura de celulases comerciais aumentando significativamente a liberação de glicose a partir do BCA, o que mostrou a capacidade das hemicelulases de melhorar a digestibilidade de celulose e tornar o processo de hidrólise mais eficiente. Nossos resultados mostram o potencial de atividades acessórias para aumentar a hidrólise enzimática, tais como XYNs, ABFs e XYLs, que estão ausentes ou presentes em pequenas proporções nas celulases comerciais. Lignocellulose is the main component of plant biomass, which consists of cellulose, hemicellulose and lignin. Xylan is the main component of hemicellulose and for its complete degradation is required cooperative action of a system consisting of several enzymes including endo-xylanases (XYN), β- xylosidases (XYL) and α-L-arabinofuranosidases (ABF). In the present work, the production of recombinant hemicellulases and its application in the hydrolysis of sugarcane bagasse (SCB) pretreated by steam explosion was performed. The first enzymes produced were two XYLs from Humicola grisea var thermoidea and this was the first study to report the genes characterization of XYLs from H. grisea, as well its heterologous expression and biochemical characterization. The two genes, hxylA and hxylB, were identified from the H. grisea genome, with 984 bp and 1617 bp, respectively. No intron and signal peptide were found and the GH43 catalytic domain was present in both. The cDNAs of both enzymes were used to transform two strains of Pichia pastoris, GS115 and SMD1168. A production optimization of enzyme denominated HXYLA was performed by a 23 factorial design. The influence of the initial cell density, methanol concentration and nitrogen source was assessed and the most significant parameter for the HXYLA production was methanol concentration. The purified enzyme was used to carry out the enzymatic characterization as optimal pH and temperature (7.0 and 50 °C), effect of ions and chemical reagents in HXYLA activity, substrate specific and kinetic parameters. HXYLA is a bifunctional enzyme, displaying β-xylosidases and α-Larabinofuranosidases activity, also that enzyme was highly tolerant xylose with high Ki value (350 mM). In addition to β-xylosidases, a recombinant endo-xylanase (HXYN2) from H. grisea was produced and purifield from a P. pastoris clone obtained by the research group from Fungal Biotechnology Laboratory (LBF/UFG). It was also produced one α-L-arabinofuranosidases (ABF3) from Penicillium purpurogenum which had its cDNA introduced in P. pastoris and the enzyme has been successfully produced. After the production of these enzymes, a 23 factorial design was used to evaluate the best formulation containing the three recombinant hemicellulases (HXYN2, ABF3 and HXYLA) for the SCB pretreated by steam explosion hydrolysis. It was possible to formulate a mixture capable to performing the conversion of the xylan fraction in the xylose, being efficient bioconversion of SCB xylan fraction an essential step for the utilization of xylose. The best formulation containing these three recombinant hemicellulases was obtained with the lowest concentration of ABF3, and it had most significant effect exerted during SCB hydrolysis. Also, the cocktail containing recombinant hemicellulases was used to supplement a mixture of commercial cellulase and it showed significantly increasing the release of glucose from the SCB hydrolysis, which showed the ability of hemicellulases to improve the digestibility of cellulose and make the hydrolysis process more efficient. Our results showed the potential for auxiliary activities to increase the enzymatic hydrolysis, such as XYNs, ABFs and XYLs who are absent or present in small amounts in comercial cellulases.

Snake venoms are recognized as a promising source of pharmacologically active substances and are potentially useful for the development of new antimicrobial drugs. This study aimed to investigate the antimicrobial activity of the venom from the rattlesnake Crotalus durissus terrificus against several bacteria. Antibacterial activity was determined by using the plate microdilution method and the activity on the bacterial envelope structure was screened by using the crystal violet assay. The proteins in crude venom were separated by electrophoresis and characterized regarding their proteolytic activity. C. d. terrificus venom exhibited antimicrobial action against gram-positive and gram-negative bacteria. MIC values were defined for Pseudomonas aeruginosa ATCC 27853 (62.5 µg/mL), Staphylococcus aureus ATCC 25923 (125 µg/mL), and Micrococcus luteus ATCC 9341 (≤500 µg/mL). For Salmonella enterica serovar typhimurium ATCC 14028 and Corynebacterium glutamicum ATCC 13032, the decrease in bacterial growth was not detected visually, but was statistically significant. The crystal violet assay demonstrated that the crude venom increased bacterial cell permeability and the secreted protein profile agreed with previous reports. The results suggest that the proteins with lytic activity against bacteria in C. d. terrificus venom deserve further characterization as they may offer reinforcements to the weak therapeutic arsenal used to fight microbial multidrug resistance.

Humicola grisea var. thermoidea is a deuteromycete that secretes a large spectrum of hydrolytic enzymes when grown on lignocellulosic residues. Agricultural residues are rich in lignocellulosic material and can be used as an alternate source for the production of different value-added products. The fungus was grown on sugarcane bagasse (SCB), wheat brain (WB), rice straw (RS) and corn cob (CC) as carbon source. The major endoxylanase, cellobiohydrolase, arabinofuranosidase and avicelase activities were observed when the fungus had grown on WB, while the major endoglucanase, FPAse and β -xylosidase activities on SCB. Secreted proteins by H. grisea upon growth on WB (HgWB) and SCB (HgSCB) by 144 h were analyzed by SDS/PAGE and Zymogram. The results suggested that HgWB and HgSCB presented a different pool of cellulases and xylanases. Cellulases and xylanases are used in enzyme cocktail to cellulose hydrolysis in cellulosic ethanol plants from SCB, hemicelluloses currently represents the largest polysaccharide fraction wasted in these plants. H. grisea enzymes (HgWB and HgSCB) and recombinant hemicellulases (endoxylanase GH11 – HXYN2, β -xylosidase – HXYLA and arabinofuranosidase ABF3) were employed to design hemicellulases cocktail which were employed to recovery arabinoxylan (AX) from steam explosion sugarcane bagasse (seSCB). The hydrolysis of seSCB with hemicellulases cocktail proved to be efficient and the release of reducing sugars (RS) from AX arriving at 91% conversion and the highest hydrolysis yields were obtained when using 4.200 U g − 1 (Units per gram substrate) of HXYN2, 10 U g − 1 of HXYLA and 4 U g − 1 of ABF3. After AX hydrolysis, the solid fraction containing cellulose was hydrolyzed with commercial cellulases and presented 28% increase in hydrolysis yield (glucose production) comparing to seSCB hydrolysis without enzymatic pre-treatment. A scanning electron microscope (SEM) was an important tool to observe the modifications in seSCB after steam explosion treatment and hydrolysis. SEM images revealed that steam explosion caused considerable damage to the fibers, such as the relaxation, fragmentation and peeling of the superficial layers of the cellulose and after enzymatic hydrolysis can be observed a greater number of cracks and more brittle in the structure. The results demonstrated that enzymatic pre-treatment with hemicellulases cocktail allowed recovery of pentoses from AX which can be converted in diverse products and improved glucose production from cellulose fraction.

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), β-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 β-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.

The dimorphic fungi Paracoccidioides spp. are the etiological agents of paracoccidioidomycosis (PCM), a prevalent systemic mycosis in Latin America. The Paracoccidioides lutzii response to oxidative stress is largely unexplored. Thioredoxins (TRX) are involved in the regulation of the redox environment in the cell, responding to oxidative stress in several organisms. In this study, we describe the isolation and characterization of a cDNA encoding a thioredoxin 1 from yeast cells from P. lutzii. The cDNA codes for a 12kDa protein containing the characteristic thioredoxin active site. The thioredoxin 1 gene was expressed in Escherichia coli and the isolated thioredoxin 1 recombinant protein as the native PlTRX1 from yeast cells showed insulin reduction activity in vitro. We also showed by semi-quantitative RT-PCR analysis that the expression of thioredoxin 1 gene was induced in response to H2O2 and may exert an antioxidant activity in vivo. Our results suggest that the thioredoxin 1 may play an important role in controlling the redox status in P. lutzii which may contribute to this organism's virulence.

One full-length β-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 β-xylosidase and α-arabinfuranosidase activities. The Km and Vmax values were 1.3mM and 39.1U/mg, respectively, against 4-nitrophenyl β-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 β-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.

Xylanases (EC 3.2.1.8) are hydrolytic enzymes, which randomly cleave the β-1,4-linked xylose residues from xylan. The synthetic gene xynBS27 from Streptomyces sp. S27 was successfully cloned and expressed in Pichia pastoris. The full-length gene consists of 729 bp and encodes 243 amino acids including 51 residues of a putative signal peptide. This enzyme was purified in two steps and was shown to have a molecular weight of 20 kDa. The purified r-XynBS27 was active against beechwood xylan and oat spelt xylan as expected for GH 11 family. The optimum pH and temperature values for the enzyme were 6.0 and 75 °C, respectively. The Km and Vmax were 12.38 mg/mL and 13.68 μmol min/mg, respectively. The r-XynBS27 showed high xylose tolerance and was inhibited by some metal ions and by SDS. r-XynBS27 was employed as an additive in the bread making process. A decrease in firmness, stiffness and consistency, and improvements in specific volume and reducing sugar content were recorded.

portuguesA expressao genica de ErbB1 e ErbB2 e a imunomarcacao de EGFR (Her1) e Her2 (c-erbB-2) foram avaliadas para verificar o envolvimento desses receptores em lesoes pre-malignas e malignas da prostata canina, como a atrofia proliferativa inflamatoria (PIA) e o carcinoma prostatico (PC). Em relacao a intensidade de imunomarcacao para EGFR, nao houve diferenca entre o tecido prostatico normal e com PIA e PC. Em relacao a Her2, observou-se diferenca de imunomarcacao entre o tecido prostatico normal e aqueles com PIA e PC e entre os com PIA e PC. Nao houve correlacao entre EGFR e Her2. O gene ErbB1 foi detectado em duas amostras normais, uma de PIA e em todas as amostras de PC. O gene ErbB2 foi detectado em duas amostras de PIA e em todas as amostras de PC, nao sendo detectado no tecido prostatico normal. Conclui-se que EGFR e Her2 atuam nas lesoes de PIA e PC, sugerindo o envolvimento destes na carcinogenese da prostata canina. Palavras-chave: receptor de fator de crescimento; Her1; c-erbB2; PIA; RT-PCR EnglishGene expression of ErbB1 and ErbB2, and immunostaining of EGFR (Her1) and Her2 (c-erbB-2) were evaluated in this study to ascertain whether these receptors are involved in the evolution of canine premalignant and malignant prostatic lesions, as proliferative inflammatory atrophy (PIA) and prostatic carcinoma (PC). With regards to the intensity of EGFR immunostaining, there was no difference between normal prostatic tissue and tissues with PIA or PC. In relation to Her2 immunostaining, there were differences between normal prostatic tissue and those with PIA and PC, as also differences between prostates with PIA and PC. There was no correlation between EGFR and Her2 immunostaining. ErbB1 gene product was detected in two normal tissue samples, in one with PIA, and in all samples with PC. ErbB2 mRNA was recorded in two canine samples with PIA, in all with PC, but was not detected in normal prostatic tissue. It was concluded that EGFR and Her2 play roles in canine PIA and PC, suggesting that those receptors may be involved in canine prostatic carcinogenesis. Key words: growth factor receptor; Her1; c-erbB-2; PIA; RT-PCR