An acid phosphatase from Trichoderma harzianum was purified in a single step using a phenyl-Sepharose chromatography column. A typical procedure showed 22-fold purification with 56% yield. The purified enzyme showed as a single band on SDS-PAGE with an apparent molecular weight of 57.8 kDa. The pH optimum was 4.8 and maximum activity was obtained at 55 degrees C. The enzyme retained 60% of its activity after incubation at 55 degrees C for 60 min. The K (m) and V (max) values for p-nitrophenyl phosphate (p-NPP) as a substrate were 165 nM and 237 nM min(-1), respectively. The enzyme was partially inhibited by inorganic phosphate and strongly inhibited by tungstate. Broad substrate specificity was observed with significant activities for p-NPP, ATP, ADP, AMP, fructose 6-phosphate, glucose 1-phosphate and phenyl phosphate.

The plant cell wall is a source of fermentable sugars in second-generation bioethanol production. However, cellulosic biomass hydrolysis remains an obstacle to bioethanol production in an efficient and low-cost process. Clostridium thermocellum has been studied as a model organism able to produce enzymatic blends that efficiently degrade lignocellulosic biomass, and also as a fermentative microorganism in a consolidated process for the conversion of lignocellulose to bioethanol. In this study, a C. thermocellum strain (designated B8) isolated from goat rumen was characterized for its ability to grow on sugarcane straw and cotton waste, and to produce cellulosomes. We also evaluated C. thermocellum gene expression control in the presence of complex lignocellulosic biomasses. This isolate is capable of growing in the presence of microcrystalline cellulose, sugarcane straw and cotton waste as carbon sources, producing free enzymes and residual substrate-bound proteins (RSBP). The highest growth rate and cellulase/xylanase production were detected at pH 7.0 and 60 °C, after 48 h. Moreover, this strain showed different expression levels of transcripts encoding cellulosomal proteins and proteins with a role in fermentation and catabolic repression.

Acid phosphatases (ACPases) are produced by a variety of fungi and have gained attention due their biotechnological potential in industrial, diagnosis and bioremediation processes. These enzymes play a specific role in scavenging, mobilization and acquisition of phosphate, enhancing soil fertility and plant growth. In this study, a new ACPase from Trichoderma harzianum, named ACPase II, was purified and characterized as a glycoprotein belonging to the acid phosphatase family. ACPase II presents an optimum pH and temperature of 3.8 and 65°C, respectively, and is stable at 55°C for 120 min, retaining 60% of its activity. The enzyme did not require metal divalent ions, but was inhibited by inorganic phosphate and tungstate. Affinity for several phosphate substrates was observed, including phytate, which is the major component of phosphorus in plant foods. The inhibition of ACPase II by tungstate and phosphate at different pH values is consistent with the inability of the substrate to occupy its active site due to electrostatic contacts that promote conformational changes, as indicated by fluorescence spectroscopy. A higher affinity for tungstate rather than phosphate at pH 4.0was observed, in accordance with its highest inhibitory effect. Results indicate considerable biotechnological potential of the ACPase II in soil environments.

A Deus por me dar o suporte necessário para ir sempre em frente nos momentos difíceis e por todas as benções realizadas ao longo desses anos. Aos meus pais, Aparecida e Zenon, por todo o esforço em me proporcionar uma educação de qualidade e serem um exemplo de caráter, princípio e honestidade. Obrigada por todo incentivo, amor e carinho. Às minhas irmãs, Danielle e Raianne por estarem ao meu lado em todos os momentos, pelos conselhos, pelas risadas, por todo o cuidado e complicidade. Ao meu esposo Humberto, pelo companheirismo, por acreditar no meu potencial e me alegrar com uma palavra de incentivo nos momentos mais difíceis e entender a minha ausênsia para a realização de algumas etapas do trabalho. À minha filha Maria Luísa que mesmo tão pequena já me ensina tanto e me transforma em uma pessoa melhor. Amo muito vocês! Ao meu orientador Dr. Carlos Roberto Felix, por acreditar em mim e me motivar desde o início das atividades desse projeto, pela paciência, atenção e educação demonstrada em todas as nossas reuniões. Ficam o meu respeito e admiração! Ao meu co-orientador Dr. Cirano José Ulhoa, por todo apoio e ensinamentos desde o início da minha formação no mestrado e por ter-me incentivado a continuar o aperfeiçoamento dos meus estudos e proporcionar a realização desse projeto com os demais professores. Minha amizade e admiração! A professora Drª Eliane Noronha pelos ensinamentos e parceria em todas as etapas do trabalho, principalmente na finalização. Sua ajuda foi fundamental. Meus sinceros agradecimentos! Aos meus colegas e amigos de bancada do laboratório de enzimologia da