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URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Hydrolysis of galacto-oligosaccharides in soy molasses by α -galactosidases and invertase from Aspergillus terreus(Brazilian Archives of Biology and Technology, 2010-05) Reis, Angélica Pataro; Guimarães, Valéria Monteze; Ferreira, Joana Gasperazzo; Queiroz, José Humberto de; Oliveira, Maria Goreti Almeida; Falkoski, Daniel Luciano; Almeida, Maíra Nicolau de; Rezende, Sebastião Tavares deTwo α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ºC were partially purified by gel filtration and hydrophobic interaction chromatographies. Optimum pH and temperatures for P1, P2 and invertase preparations were 4.5-5.0, 5.5 and 4.0 and 60, 55 and 65ºC, respectively. The KM app for ρ -nitrophenyl-α -D-galactopyranoside were 1.32 mM and 0.72 mM for P1 and P2, respectively, while the KM app value for invertase, using sacarose as a substrate was 15.66 mM. Enzyme preparations P1 and P2 maintained their activities after pre-incubation for 3 h at 50ºC and invertase maintained about 90% after 6 h at 55 ºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.Item Avaliação da concentração de proteínas e da atividade de α-galactosidases nos cotilédones e no eixo embrionário de sementes de Dalbergia nigra durante a germinação(Acta Amazonica, 2011) Carrijo, Lanna Clicia; Borges, Eduardo Euclydes de Lima e; Rezende, Sebastião Tavares de; Pontes, Cláudia Aparecida; Silva, Aderlan Gomes da; Lopes, Mariana RochaEste trabalho teve como objetivos a quantificação de proteínas e da atividade da enzima α-galactosidase, no eixo embrionário e nos cotilédones, de sementes de Dalbergia nigra (jacarandá-da-bahia) durante a germinação. As sementes foram colocadas para embeber em água por sete dias, sendo retiradas amostras para a avaliação bioquímica e cinética da enzima. A atividade da enzima α-galactosidase aumenta com a embebição das sementes nos dois compartimentos, embora não esteja presente no eixo embrionário de sementes secas. A diferença na atividade da enzima entre os cotilédones e o eixo embrionário foi significativa. O pH 5,5 foi o de máxima atividade para as enzimas de ambos os compartimentos. A temperatura que mais estimulou a atividade da enzima nos cotilédones foi 50 ºC e de 50 a 60 ºC no eixo embrionário. A atividade da α-galactosidase foi inibida por β-mercaptoetanol e cobre, em ambos os compartimentos, enquanto a lactose e o cloreto de sódio estimularam a atividade tanto nos cotilédones como no eixo embrionário. Os valores de KM para enzimas do eixo embrionário e dos cotilédones foram de 0,239 e 0,228 mM, respectivamente.Item Propriedades enzimáticas da enzima ALS de Cyperus difformis e mecanismo de resistência da espécie ao herbicida pyrazosulfuron-ethyl(Ciência Rural, 2010-12) Magro, Taísa Dal; Rezende, Sebastião Tavares de; Agostinetto, Dirceu; Vargas, Leandro; Silva, Antônio Alberto da; Falkoski, Daniel LucianoCyperus difformis L. é uma planta daninha ocorrente em lavouras de arroz irrigado, que tem apresentado dificuldade de controle devido à resistência a herbicidas inibidores da enzima ALS. Os objetivos deste trabalho foram investigar características cinéticas da enzima ALS de biótipos de C. difformis e determinar as bases bioquímicas da resistência da espécie ao herbicida pyrazosulfuron-ethyl. Para isso, foram conduzidos experimentos em laboratório do BIOAGRO/UFV. O método utilizado baseou-se na metologia utilizada por CAREY et al. (1997) e adaptada por VARGAS et al. (1999), com algumas modificações. Foram avaliadas a concentração de substrato (piruvato) que fornece velocidade inicial igual à metade da velocidade máxima de reação (K M ) e velocidade máxima de reação (V máx ), bem como a atividade da enzima ALS na presença do inibidor (pyrazosulfuron-ethyl). Diante dos resultados, pode-se observar que a resistência de C. difformis a pyrazosulfuron-ethyl é decorrente da insensibilidade da enzima ALS ao herbicida, não acarretando, porém, prejuízo aos parâmetros cinéticos K M e V máx da enzima ALS.Item Thermostability improvement of Orpinomyces sp. xylanase by directed evolution(Journal of Molecular Catalysis B: Enzymatic, 2012-09) Trevizano, Larissa Mattos; Ventorim, Rafaela Zandonade; Rezende, Sebastião Tavares de; Silva Junior, Floriano Paes; Guimarães, Valéria MontezeThe methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.Item Removal of oligosaccharides in soybean flour and nutritional effects in rats(Food Chemistry, 2010-01-15) Brasil, Ana Paula Rodrigues; Rezende, Sebastião Tavares de; Pelúzio, Maria do Carmo Gouveia; Guimarães, Valéria MontezeThe objectives of this work were to establish a safe and economically viable process for the removal of raffinose oligosaccharides (RO) from soy flour and compare the effects of RO elimination from diets with regard to nutritional parameters by testing in Wistar rats. Debaryomyces hansenii UFV-1 was cultivated in suspension of defatted soy flour (1:10 w/v). An increase in α-galactosidase activity was observed in the medium, with a consequent decrease in the RO concentration. A total reduction of RO was achieved at 36 h of incubation. The diet containing soy flour free of RO presented higher digestibility, 91.28%, in relation to the diet containing soy flour with RO, 87.14%. However, the removal of the oligosaccharides from the diet did not promote a significant improvement in the values of weight gain, and other nutritional parameters tested on rats, during the experimental period of 14 days.Item Purification and characterization of Aspergillus terreus α-Galactosidases and their use for hydrolysis of Soymilk Oligosaccharides(Applied Biochemistry and Biotechnology, 2011-02-18) Ferreira, Joana Gasperazzo; Reis, Angélica Pataro; Guimarães, Valéria Monteze; Falkoski, Daniel Luciano; Silva Fialho, Lílian da; Rezende, Sebastião Tavares deα-Galactosidases has the potential to hydrolyze α-1-6 linkages in raffinose family oligosaccharides (RFO). Aspergillus terreus cells cultivated on wheat bran produced three extracellular forms of α-galactosidases (E1, E2, and E3). E1 and E2 α-galactosidases presented maximal activities at pH 5, while E3 α-galactosidase was more active at pH 5.5. The E1 and E2 enzymes showed stability for 6 h at pH 4–7. Maximal activities were determined at 60, 55, and 50°C, for E1, E2, and E3 α-galactosidase, respectively. E2 α-galactosidase retained 90% of its initial activity after 70 h at 50°C. The enzymes hydrolyzed ρNPGal, melibiose, raffinose and stachyose, and E1 and E2 enzymes were able to hydrolyze guar gum and locust bean gum substrates. E1 and E3 α-galactosidases were completely inhibited by Hg2+, Ag+, and Cu2+. The treatment of RFO present in soy milk with the enzymes showed that E1 α-galactosidase reduced the stachyose content to zero after 12 h of reaction, while E2 promoted total hydrolysis of raffinose. The complete removal of the oligosaccharides in soy milk could be reached by synergistic action of both enzymesItem Cellulases and hemicellulases from endophytic acremonium species and its application on sugarcane bagasse hydrolysis(Appl Biochem Biotechnol, 2011-05-02) Almeida, Maíra Nicolau de; Guimarães, Valéria Monteze; Bischoff, Kenneth M.; Falkoski, Daniel Luciano; Pereira, Olinto Liparini; Gonçalves, Dayelle S. P. O.; Rezende, Sebastião Tavares deThe aim of this work was to have cellulase activity and hemicellulase activity screenings of endophyte Acremonium species (Acremonium zeae EA0802 and Acremonium sp. EA0810). Both fungi were cultivated in submerged culture (SC) containing L -arabinose, D -xylose, oat spelt xylan, sugarcane bagasse, or corn straw as carbon source. In solid-state fermentation, it was tested as carbon source sugarcane bagasse or corn straw. The highest FPase, endoglucanase, and xylanase activities were produced by Acremonium sp. EA0810 cultivated in SC containing sugarcane bagasse as a carbon source. The highest β-glucosidase activity was produced by Acremonium sp. EA0810 cultivated in SC using D -xylose as carbon source. A. zeae EA0802 has highest α-arabinofuranosidase and α-galactosidase activities in SC using xylan as a carbon source. FPase, endoglucanase, β-glucosidase, and xylanase from Acremonium sp. EA0810 has optimum pH and temperatures of 6.0, 55 °C; 5.0, 70 °C; 4.5, 60 °C; and 6.5, 50 °C, respectively. α-Arabinofuranosidase and α-galactosidase from A. zeae EA0802 has optimum pH and temperatures of 5.0, 60 °C and 4.5, 45 °C, respectively. It was analyzed the application of Acremonium sp. EA0810 to hydrolyze sugarcane bagasse, and it was achieved 63% of conversion into reducing sugar and 42% of conversion into glucose.