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URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
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Item Purification and characterization of an a-galactosidase from Aspergillus fumigatus(Brazilian Archives of Biology and Technology, 2005-03) Rezende, Sebastião Tavares de; Guimarães, Valéria Monteze; Rodrigues, Marília de Castro; Felix, Carlos RobertoAspergillus fumigatus secreted invertase (b-fructofuranosidase) and a-galactosidase enzymatic activities able to hydrolyzing raffinose oligosaccharides (RO). a-Galactosidase was induced by galactose, melibiose and raffinose, but galactose was the most efficient inducer. It was purified by gel filtration and two ion exchange chromatographies and showed Mw of 54.7 kDa. The purified enzyme showed maximal activity against p-nitrophenyl-a-D-galactopyranoside (pNPGal) at pH 4.5-5.5 and 55 °C, and retained about 80% of the original activity after incubation for 90 minutes at 50ºC. The KM for pNPGal was 0.3 mM. Melibiose was hydrolyzed by the enzyme but raffinose was very poor substrate.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 Hydrolysis of oligosaccharides in soybean flour by soybean α-galactosidase(Food Chemistry, 2015-12) Guimarães, Valéria Monteze; José, Inês Chamel; Oliveira, Maria Goreti de Almeida e; Oliveira, Maria Goreti de Almeida e; Barros, Everaldo Gonçalves de; Moreira, Maurílio Alves; Viana, Simone de Fátima; Rezende, Sebastião Tavares deRaffinose oligosaccharides (ROs) make up a substantial part (40%) of the soluble sugars found in soybean seeds and are responsible for flatulence after the ingestion of soybean and other legumes. Consequently, soy-based foods would find a broader approval if the ROs were removed from soybean products or hydrolysed by α-galactosidases. During soybean seed germination, of content the ROs decrease substantially, while the α-galactosidase activity increases. α-Galactosidase was partially purified from germinating seeds by partition in an aqueous two-phase system and ion-exchange chromatography. The enzyme preparation presented maximal activities against ρ-nitrophenyl-α-d-galactopyranoside (ρNPGal) at 60 °C and a pH of 5.0 and the KM app values for ρNPGal, melibiose, and raffinose of the enzyme preparation were 0.33, 0.42, and 6.01 mM, respectively. The enzyme was highly inhibited by SDS, copper, and galactose. Hydrolysis of soybean flour ROs by enzyme preparation reduced the stachyose and raffinose contents by 72.3% and 89.2%, respectively, after incubation for 6 h at 40 °C.Item Treatment of soy milk with Debaryomyces hansenii cells immobilised in alginate(Food Chemistry, 2009-05-15) Souza Júnior, Waldeck Campanha de; Rezende, Sebastião Tavares de; Viana, Pollyanna Amaral; Falkoski, Daniel Luciano; Reis, Angélica Pataro; Machado, Solimar Gonçalves; Barros, Everaldo Gonçalves de; Guimarães, Valéria MontezeWhole cells of Debaryomyces hansenii UFV-1 were permeabilised with ethanol and immobilised in calcium alginate hydrogel. The optimum pH and temperature for α-galactosidase activities of permeabilised free (PFC) and permeabilised immobilised cells (PIC) were 4.5 and 60 °C; and 4.0 and 70 °C, respectively. PIC α-galactosidase was more stable than that of PFC. The incubation of PIC at 60 and 70 °C promoted an increase in α-galactosidase activity. The α-galactosidase activity was maintained when PIC was used in three repeated batches. The Km values for PIC and PFC α-galactosidases, with ρNPαGal, were 0.82 mM and 0.30 mM, respectively. Soy milk treatment with PIC for 6 h at 60 °C promoted 100% hydrolysis of raffinose oligosaccharides.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 xylanases from the fungus Chrysoporthe cubensis for production of xylooligosaccharides and fermentable sugars(Applied Biochemistry and Biotechnology, 2016-12-24) Sousa Gomes, Kamila de; Maitan-Alfenas, Gabriela P.; Andrade, Lorena G. A. de; Falkoski, Daniel Luciano; Guimarães, Valéria Monteze; Alfenas, Acelino C.; Rezende, Sebastião Tavares deXylanases from the pathogen fungus Chrysoporthe cubensis were produced under solid state fermentation (SSF) using wheat bran as carbon source. The enzymatic extracts were submitted to ion exchange (Q Sepharose) and gel filtration chromatography methods (Sephadex S-200) for purification. The xylanases were divided into three groups: P1 showed better performance at 60 °C and pH 4.0, P2 at 55 °C and pH 3.0, and P3 at 80 °C and pH 3.0. Oat spelt xylan was the best substrate hydrolyzed by P1 and P3, while beechwood xylan was better degraded by P2. Carboxymethyl cellulose (CMC) and p-nitrophenyl-β-d-xylopyranoside (p-NPβXyl) were not hydrolyzed by any of the xylanases. The K M ’ or K M values, using oat spelt xylan as substrate, were 2.65 mg/mL for P1, 1.81 mg/mL for P2, and 1.18 mg/mL for P3. Xylobiose and xylotriose were the main xylooligosaccharides of oat spelt xylan degradation, indicating that the xylanases act as endo-β-1,4-xylanases. Xylanases also proved to be efficient for hydrolysis of sugarcane bagasse when used as supplement of a commercial cocktail due to the increase of the reducing sugar release.Item Covalent immobilization of α-Galactosidase from Penicillium griseoroseum and its application in Oligosaccharides Hydrolysis(Applied Biochemistry and Biotechnology, 2008-10-21) Falkoski, Daniel Luciano; Guimarães, Valéria Monteze; Queiroz, Marisa Vieira de; Araújo, Elza Fernandes de; Almeida, Maíra Nicolau de; Barros, Everaldo Gonçalves de; Rezende, Sebastião Tavares dePartially purified α-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized α-galactosidase showed optimal activity at 45 °C and pH values of 5 and 4, respectively. Immobilized α-galactosidase was more stable at higher temperatures and pH values. Immobilized α-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 °C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized α-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized α-galactosidase respectively. Immobilized α-galactosidase was reutilized eight times without any decrease in its activity.