Bioquímica e Biologia Molecular

URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11837

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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 de
Two α -galactosidase (P1 and P2) and one invertase present in the culture of Aspergillus terreus grown on wheat straw for 168 h at 28ÂºC 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 65ÂºC, 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 50ÂºC and invertase maintained about 90% after 6 h at 55 ÂºC. P1 and P2 presented different inhibition sensitivities by Ag+, D-galactose, and SDS. All enzyme preparations hydrolyzed galacto-ologosaccharides present in soymolasses.
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 de
Raffinose 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.
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 Monteze
The 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.
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 Monteze
The 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.
Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2
(International Journal of Biological Macromolecules, 2017-08-03) Ventorim, Rafaela Zandonade; Mendes, Tiago Antônio de Oliveira; Trevizano, Larissa Mattos; Camargos, Ana Maria dos Santos; Guimarães, Valéria Monteze
Xylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme con- structs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8 U mg^−1 of protein, respec- tively, while the WT activity was 444.1 U mg^−1 of protein. WT, SWT and SM2 showed half-life values at 50 ◦ C of 0.8, 2.3 and 29.5 h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.
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 de
Xylanases 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.
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 de
α-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 enzymes
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 de
The 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.
Increased enzymatic hydrolysis of sugarcane bagasse from enzyme recycling
(Biotechnology for Biofuels, 2015-01-22) Visser, Evan Michael; Leal, Tiago Ferreira; Almeida, Maíra Nicolau de; Guimarães, Valéria Monteze
Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings. It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes. Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity.
The influence of pretreatment methods on saccharification of sugarcane bagasse by an enzyme extract from Chrysoporthe cubensis and commercial cocktails: A comparative study
(Bioresource Technology, 2015-06-14) Maitan-Alfenas, Gabriela Piccolo; Visser, Evan Michael; Alfenas, Rafael Ferreira; Nogueira, Bráulio Ris G.; Campos, Guilherme Galvão de; Milagres, Adriane Ferreira; Vries, Ronald P. de; Guimarães, Valéria Monteze
Biomass enzymatic hydrolysis depends on the pretreatment methods employed, the composition of initial feedstock and the enzyme cocktail used to release sugars for subsequent fermentation into ethanol. In this study, sugarcane bagasse was pretreated with 1% H2SO4 and 1% NaOH and the biomass saccharification was performed with 8% solids loading using 10 FPase units/g of bagasse of the enzymatic extract from Chrysoporthe cubensis and three commercial cocktails for a comparative study. Overall, the best glucose and xylose release was obtained from alkaline pretreated sugarcane bagasse. The C. cubensis extract promoted higher release of glucose (5.32 g/L) and xylose (9.00 g/L) than the commercial mixtures. Moreover, the C. cubensis extract presented high specific enzyme activities when compared to commercial cocktails mainly concerning to endoglucanase (331.84 U/mg of protein), β-glucosidase (29.48 U/mg of protein), β-xylosidase (2.95 U/mg of protein), pectinase (127.46 U/mg of protein) and laccase (2.49 U/mg of protein).

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