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Tatiana Souza Porto - One of the best experts on this subject based on the ideXlab platform.
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extraction and purification of Aspergillus tamarii β fructofuranosidase with transfructosylating activity using aqueous biphasic systems peg phosphate and magnetic field
Preparative Biochemistry & Biotechnology, 2021Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Camila Souza Porto, Maria Itais Dos Santos Bernardino, Talis Bruno Santos Silva, Tatiana Souza PortoAbstract:β-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.
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production extraction and thermodynamics protease partitioning from Aspergillus tamarii kita ucp1279 using peg sodium citrate aqueous two phase systems
Preparative Biochemistry & Biotechnology, 2020Co-Authors: Yuri Matheus Silva Amaral, Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Tatiana Souza PortoAbstract:The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyet...
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extraction of protease from Aspergillus tamarii urm 4634 in aqueous two phase system under continuous and discontinuous process
Preparative Biochemistry & Biotechnology, 2020Co-Authors: J G W Siqueira, T M S Torres, B M V Alves, Ana Lucia Figueiredo Porto, Tatiana Souza PortoAbstract:Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene g...
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biochemical characterization and kinetic thermodynamic study of Aspergillus tamarii urm4634 β fructofuranosidase with transfructosylating activity
Biotechnology Progress, 2019Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Marcos Fellipe Da Silva, Tatiana Souza PortoAbstract:This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.
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biophysical photochemical and biochemical characterization of a protease from Aspergillus tamarii urm4634
International Journal of Biological Macromolecules, 2018Co-Authors: Osmar Soares Da Silva, Elizane Melo De Almeida, Jonatas De Carvalho Silva, Flavia Sousa, Odete Goncalves, Bruno Sarmento, Maria Teresa Nevespetersen, Tatiana Souza PortoAbstract:Abstract Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N–formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (T m ) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed T m values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest T m was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. The acquired data permits to select the best physicochemical parameters to secure the optimal activity and stability when used in biotechnological applications. Furthermore, the conformal changes induced by temperature in the protein are directly correlated with its level of biochemical activity.
Osmar Soares Da Silva - One of the best experts on this subject based on the ideXlab platform.
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production extraction and thermodynamics protease partitioning from Aspergillus tamarii kita ucp1279 using peg sodium citrate aqueous two phase systems
Preparative Biochemistry & Biotechnology, 2020Co-Authors: Yuri Matheus Silva Amaral, Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Tatiana Souza PortoAbstract:The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyet...
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biophysical photochemical and biochemical characterization of a protease from Aspergillus tamarii urm4634
International Journal of Biological Macromolecules, 2018Co-Authors: Osmar Soares Da Silva, Elizane Melo De Almeida, Jonatas De Carvalho Silva, Flavia Sousa, Odete Goncalves, Bruno Sarmento, Maria Teresa Nevespetersen, Tatiana Souza PortoAbstract:Abstract Circular dichroism (CD) and fluorescence spectroscopy (FS) were used to monitor the pH-dependent conformational and structural stability changes induced by temperature and UV light on the protease from Aspergillus tamarii URM4634 at different pH values. The formation of photoproducts, such as N–formylkynurenine, dityrosine and kynurenine, were monitored with FS. The pH-dependent melting temperatures (T m ) were determined using CD and FS from 20 to 90 °C. Conformational changes were correlated with the pH-dependent biochemical activities. CD revealed that the protease is rich in α-helices. Thermal denaturation was irreversible at all pH range and displayed T m values from 42.8 to 67.8 °C (CD) and from 38 to 60.3 °C (FS), which the highest T m was observed at pH 6. The light and temperature induced to the formation of photoproducts was more intense at high pH value. Despite the biochemical data shows optimum pH 9, the highest stability was at pH 6, maintaining 100% of activity after 24 h. The acquired data permits to select the best physicochemical parameters to secure the optimal activity and stability when used in biotechnological applications. Furthermore, the conformal changes induced by temperature in the protein are directly correlated with its level of biochemical activity.
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purification and characterization of a novel extracellular serine protease with collagenolytic activity from Aspergillus tamarii urm4634
International Journal of Biological Macromolecules, 2018Co-Authors: Osmar Soares Da Silva, Elizane Melo De Almeida, Allan Henrique Felix De Melo, Tatiana Souza PortoAbstract:Abstract An extracellular serine-protease from Aspergillus tamarii URM4634 was purified and characterized. The possibility of using Aspergillus tamarii URM4634 protease in detergent formulations and collagenolytic activity was investigated. The protease demonstrated excellent stability at pH range 7.0–11.0, the optimum being at pH 9.0. The enzyme was stable at 40 °C for 180 min, enhanced by Mg++ and Ca++, but inhibited by Zn++, and strongly inhibited by phenylmethylsulfonyl fluoride (PMSF), suggested as serine-protease. The azocasein substrate result showed Km = 0.434 mg/mL and Vmax = 7.739 mg/mL/min. SDS-PAGE and azocasein zymography showed that the purified alkaline protease (2983.8 U/mg) had a molecular mass of 49.3 kDa. The enzyme was purified by column chromatography using Sephadex A50 resin. The proteolytic activity was activated by SDS (sodium dodecyl sulfate), Tween-80, Tween 20 and Triton-100. This study demonstrated that A. tamarii URM4634 protease has potent, stable and compatible collagenolytic activity to the desired level in local laundry detergent brands compared with similar enzymes produced by solid-state fermentation. This protease can thus be chosen as an option in both the food industry to tenderization meat and the detergent industry to washing process.
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peg sodium citrate aqueous two phase systems to in situ recovery of protease from Aspergillus tamarii urm4634 by extractive fermentation
Biocatalysis and agricultural biotechnology, 2018Co-Authors: Osmar Soares Da Silva, Raniele Oliveira Alves, Tatiana Souza PortoAbstract:Abstract This paper investigates the influence of polyethylene glycol (PEG) molar mass, concentrations of PEG and sodium citrate, and pH, in situ recovery on protease produced by Aspergillus tamarii URM4634. A biochemical characterization study was conducted, and thermodynamic parameters were determined in the test which used 8000 (g/mol) PEG molar mass, PEG concentration 24% (w/w), sodium citrate concentration 20% (w/w) and pH 6.0 with partition coefficient of 35.0, activity yield of 98.4 and concentration factor of 2.14. This PEG-phase had an optimum pH and temperature of 8.0 and 60 °C, respectively, being inhibited by EDTA (83.3%) which indicated that this's a metalloprotease. The thermodynamic studies of the protease had an activation energy of 19.01 kJ/mol and a deactivation energy of 29.6 kJ/mol. It was in this range that the following estimates could be made: enthalpy of ΔH*d 29.7 kJ/mol, entropy of ΔS*d − 265.0 kJ/mol K and Gibbs free energy of 120.0 ≤ ΔG*d ≤ 125.9 kJ/mol. These results show a possible scaling-up of the bioreactor process which, under the conditions in the shaker flask, showed considerable values in the integrated production and extraction process, thus demonstrating the potential of this process for obtaining a high recovery of protease and similar products.
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purificacao de proteases de Aspergillus tamarii urm4634 por cromatografia de troca ionica
Revista Brasileira de Agrotecnologia, 2017Co-Authors: Raniele Oliveira Alves, Osmar Soares Da Silva, Matheus Henrique Gouveia Gomes, Tatiana Souza PortoAbstract:Proteases sao enzimas que catalisam reacoes hidroliticas onde ha a quebra das ligacoes peptidicas entre os aminoacidos das proteinas. Sua obtencao por microrganismos apresenta algumas vantagens, como a facilidade de producao em larga escala, condicoes controladas de temperatura e pH, bem como vem sendo aplicada na industria de alimentos. O presente trabalho objetivou purificar as proteases produzidas por Aspergillus tamarii URM4634 utilizando a cromatografico de troca ionica. O micro-organismo A. tamarii URM4634 se mostrou um potencial produtor de proteases, apresentando uma atividade proteasica de 60,3 U/mL apos 72h de fermentacao. Os processos de dowstream que envolvem etapas de purificacao, obtiveram resultados satisfatorios por precipitacao por acetona, e pela cromatografia de troca ionica DEAE-Sephadex A50, apresentando um aumento no seu fator de purificacao em 4,4 vezes com uma recuperacao de 24,3%. Assim, a purificacao por cromatografia de troca ionica mostrou-se vantajosa para utilizacao destas enzimas em aplicacoes na industria de alimentos.
Ana Lucia Figueiredo Porto - One of the best experts on this subject based on the ideXlab platform.
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biotechnological purification of a β fructofuranosidase β ffase from Aspergillus tamarii kita aqueous two phase system peg citrate and biochemical characterization
Biocatalysis and agricultural biotechnology, 2021Co-Authors: Juanize Matias Da Silva Batista, Romero Marcos Pedrosa Brandaocosta, Kethylen Barbara Barbosa Cardoso, Thiago Pajeu Nascimento, Wendell Wagner Campos Albuquerque, Marcia Nieves Carneiro Da Cunha, Camila Souza Porto, Raquel Pedrosa Bezerra, Ana Lucia Figueiredo PortoAbstract:Abstract β-fructofuranosidases (EC3.2.1.26) are members of the GH32 family of glycoside hydrolases, which include more than 390 enzymes of vegetable and microbial origins, used in several biotechnological applications. Thus, this research aimed to produce a β-fructofuranosidase obtained by Aspergillus tamarii through solid state fermentation, and to purify by Aqueous Two-Phase System (ATPS). Summary results presented the optimal parameters to produce the β-fructofuranosidase used wheat bran as a substrate at 30 °C for 48 h, and purification process using ATPS with polyethylene glycol and sodium citrate (PEG/sodium citrate), where the β-fructofuranosidase preferably partitioned to the salt-rich phase, the best run (24% of PEG 400, 20% sodium citrate, pH 8) which presented a higher purification factor 6.42 with 12.39 U/mL activity and 352% yield. Optimum parameter was pH 5.15 and temperature of 55 °C, respectively. The purified enzyme showed excellent thermal stability and exhibited a half-life of 60 min at 65 °C. Kinetics results for enzyme showed for Sucrose substrate the enzyme showed Km of 42.9 ± 2.21 mM and Vmax of 180.2 ± 2.8 μM min−1 mg−1 of protein. Although Vmax was the highest for 1-Kestose (219.4 ± 2.7 μM min−1 mg−1 of protein) the preferred substrate of Aspergillus tamarii β-fructofuranosidase (β-FFase) was Nystose (Km of 3.8 ± 0.15 mM). SDS-PAGE revealed a single band of protein at ~66 kDa. Finally, this study demonstrated the potential of ATPS to purify a β-fructofuranosidase with application in biotechnological field aiming to functional foods.
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purification and biochemical characterization of an extracellular fructosyltransferase rich extract produced by Aspergillus tamarii kita ucp1279
Biocatalysis and agricultural biotechnology, 2020Co-Authors: Juanize Matias Da Silva Batista, Romero Marcos Pedrosa Brandaocosta, Marcia Nieves Carneiro Da Cunha, Helio O S Rodrigues, Ana Lucia Figueiredo PortoAbstract:Abstract The market for prebiotics is gaining strength and is growing greatly in recent years since the consumers demand are changing and are highly influenced by the increasing consumption of healthier products. The fructosyltransferase (FTase) mainly produced by filamentous fungi is responsible for the synthesis of fructooligosaccharides which are widely used as a bioactive ingredient in functional foods. The objectives of this work were to select the best Aspergillus strains fructosyltransferase producer, purify FTase using ion-exchange chromatography coupled to FPLC under Superdex-G75 and to produce fructooligosaccharides. Six strains of Aspergillus sp. were studied, and A. tamarii Kita was highlighted because of the greater fructosyltransferase activity. At the production stage, the maximum activity of crude extract (1629.03 U/gds) was obtained under the following conditions: wheat bran 3.0g, sucrose concentration at 20%, 106 spore/mL, 70% moisture, 12h in the presence of light, at 30 °C for 48h. Superdex-G75 was able to purify the enzyme with high activity, and after SDS-PAGE under zymography conditions, one band of protein was observed at 89.7 kDa. The specific activity of the final purified material was 112,629.03 U/gds with purification ratio of 49 times and yield of 36%. FTase presented optimum temperature at 60 °C and pH 5.0, respectively. Together, these results indicated that the FTase of Aspergillus tamarii Kita is an excellent candidate for the industrial production of FOS.
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extraction of protease from Aspergillus tamarii urm 4634 in aqueous two phase system under continuous and discontinuous process
Preparative Biochemistry & Biotechnology, 2020Co-Authors: J G W Siqueira, T M S Torres, B M V Alves, Ana Lucia Figueiredo Porto, Tatiana Souza PortoAbstract:Aqueous two-phase systems have been studied for almost a century to separate biomolecules in harmless conditions. Proteases produced by Aspergillus tamarii URM 4634 were extracted in polyethylene g...
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partitioning and extraction protease from Aspergillus tamarii urm4634 using peg citrate aqueous two phase systems
Biocatalysis and agricultural biotechnology, 2017Co-Authors: Osmar Soares Da Silva, Ana Lucia Figueiredo Porto, Rodrigo Lira De Oliveira, Attilio Converti, Matheus Henrique Gouveia Gomes, Tatiana Souza PortoAbstract:Abstract PEG-citrate Aqueous Two-Phase Systems (ATPS) were used to recover and partially purify protease from Aspergillus tamarii URM4634 produced by Solid State Fermentation. Experiments were performed according to a 2 4 -full factorial design using PEG molar mass ( M PEG ), PEG concentration ( C PEG ), citrate concentration ( C CIT ) and pH as independent variables; and purification factor ( PF ), partition coefficient ( K ) and activity yield ( Y ) as responses. Protease showed high activity in the PEG-rich phase, also M PEG and C CIT were shown to exert positive effects on all responses. The highest purification factor (3.95) was obtained using M PEG =8000 g/mol, 24% (w/w) C PEG , 20% (w/w) C CIT at pH 8.0. Consequently, the selected ATPS proved to be efficient and can be used as a first step for pre-purification of protease from solid state fermented of A. tamarii URM4634.
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novel protease from Aspergillus tamarii urm4634 production and characterization using inexpensive agroindustrial substrates by solid state fermentation
Advances in Enzyme Research, 2016Co-Authors: Osmar Soares Da Silva, Ana Lucia Figueiredo Porto, Rodrigo Lira De Oliveira, Cristina Maria De Souzamotta, Tatiana Souza PortoAbstract:This study reports the protease production from Aspergillus tamarii using agroindustrial residues as substrate for solid-state fermentation (SSF) and biochemical characterization. The highest protease production was obtained using wheat bran as substrate at 72 h fermentation with maximum proteolytic activity of 401.42 U/mL, collagenase of 243.0 U/mL and keratinase of 19.1 U/mL. The protease exhibited KM = 18.7 mg/mL and Vmax = 28.5 mg/mL/min. The optimal pH was 8.0 and stable in a wide pH range (5.0 - 11.0) during 24 h. The optimum temperature was 40°C. The proteolytic activity was inhibited by Cu2+ (33.98%) and Hg2+ (22.69%). The enzyme was also inhibited by PMSF (65.11%), indicating that is a Serine Protease. These properties suggest that alkaline protease from A. tamarii URM4634 is suitable for application in food industries and leather processing. Additionally, the present findings opened new vistas in the utilization of wheat bran and other effective agroindustrial wastes as substrates for SSF.
Rodrigo Lira De Oliveira - One of the best experts on this subject based on the ideXlab platform.
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extraction and purification of Aspergillus tamarii β fructofuranosidase with transfructosylating activity using aqueous biphasic systems peg phosphate and magnetic field
Preparative Biochemistry & Biotechnology, 2021Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Camila Souza Porto, Maria Itais Dos Santos Bernardino, Talis Bruno Santos Silva, Tatiana Souza PortoAbstract:β-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.
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production extraction and thermodynamics protease partitioning from Aspergillus tamarii kita ucp1279 using peg sodium citrate aqueous two phase systems
Preparative Biochemistry & Biotechnology, 2020Co-Authors: Yuri Matheus Silva Amaral, Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Tatiana Souza PortoAbstract:The protease from Aspergillus tamarii Kita UCP1279 extraction by aqueous two-phase PEG-Citrate (ATPS) systems, using a factorial design 24, was investigated. Then, the variables studied were polyet...
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biochemical characterization and kinetic thermodynamic study of Aspergillus tamarii urm4634 β fructofuranosidase with transfructosylating activity
Biotechnology Progress, 2019Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Marcos Fellipe Da Silva, Tatiana Souza PortoAbstract:This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.
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thermodynamic investigation of an alkaline protease from Aspergillus tamarii urm4634 a comparative approach between crude extract and purified enzyme
International Journal of Biological Macromolecules, 2017Co-Authors: Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Jonatas De Carvalho Silva, Attilio Converti, Tatiana Souza PortoAbstract:Abstract The thermostable crude proteolytic extract and purified protease produced by Aspergillus tamarii URM4634 were investigated at different temperatures. The activity results were used to estimate the activation energy of the hydrolysis reaction catalyzed by crude extract and purified protease (E* = 34.2 and 16.2 kJ/mol) as well as the respective standard enthalpy variations of reversible enzyme unfolding (ΔH°u = 31.9 and 13.9 kJ/mol). When temperature was raised from 50 to 80 °C in residual activity tests, the specific rate constant of crude proteolytic extract thermoinactivation increased from 0.0072 to 0.0378 min−1, while that of purified protease from 0.0099 to 0.0235 min−1. These values, corresponding to half-life decreases from 96.3 to 18.3 min and from 70.0 to 29.5 min, respectively, enabled us to estimate the activation energy (E*d = 49.7 and 28.8 kJ/mol), enthalpy (ΔH*d = 47.0 and 26.1 kJ/mol), entropy (ΔS*d = −141.3 and −203.1 J/mol K) and Gibbs free energy (92.6 ≤ ΔG*d ≤ 96.6 kJ/mol and 91.8 ≤ ΔG*d ≤ 98.0 kJ/mol) of thermoinactivation. Such values suggest that this protease, which proved to be highly thermostable in both forms, could be profitably exploited in industrial applications. To the best of our knowledge, this is the first comparative study on thermodynamic parameters of a serine protease produced by Aspergillus tamarii URM4634.
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partitioning and extraction protease from Aspergillus tamarii urm4634 using peg citrate aqueous two phase systems
Biocatalysis and agricultural biotechnology, 2017Co-Authors: Osmar Soares Da Silva, Ana Lucia Figueiredo Porto, Rodrigo Lira De Oliveira, Attilio Converti, Matheus Henrique Gouveia Gomes, Tatiana Souza PortoAbstract:Abstract PEG-citrate Aqueous Two-Phase Systems (ATPS) were used to recover and partially purify protease from Aspergillus tamarii URM4634 produced by Solid State Fermentation. Experiments were performed according to a 2 4 -full factorial design using PEG molar mass ( M PEG ), PEG concentration ( C PEG ), citrate concentration ( C CIT ) and pH as independent variables; and purification factor ( PF ), partition coefficient ( K ) and activity yield ( Y ) as responses. Protease showed high activity in the PEG-rich phase, also M PEG and C CIT were shown to exert positive effects on all responses. The highest purification factor (3.95) was obtained using M PEG =8000 g/mol, 24% (w/w) C PEG , 20% (w/w) C CIT at pH 8.0. Consequently, the selected ATPS proved to be efficient and can be used as a first step for pre-purification of protease from solid state fermented of A. tamarii URM4634.
Attilio Converti - One of the best experts on this subject based on the ideXlab platform.
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extraction and purification of Aspergillus tamarii β fructofuranosidase with transfructosylating activity using aqueous biphasic systems peg phosphate and magnetic field
Preparative Biochemistry & Biotechnology, 2021Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Camila Souza Porto, Maria Itais Dos Santos Bernardino, Talis Bruno Santos Silva, Tatiana Souza PortoAbstract:β-fructofuranosidases (FFases) are enzymes involved in sucrose hydrolysis and fructo-oligosaccharides' production which are of great interest for the food industry. FFase from Aspergillus tamarii URM4634 was extracted using PEG/Phosphate Aqueous Biphasic Systems (ABS), and the impact of magnetic field on the extraction behavior was evaluated. A 24-full experimental design was employed to study the influence of molar mass of PEG, concentrations of PEG and phosphate and pH on the selected response variables, i.e., partition coefficient (K), purification factor (PF), activity yield (Y) and selectivity (S). The influence of magnetic field during partition and NaCl concentration on the same responses was also studied. The best results of FFase extraction without magnetic field (K = 0.50, PF = 4.05, Y = 72.66% and S = 0.06) were observed at pH 8.0 using 12.5% (w/w) PEG 400 and 25% (w/w) NaH2PO4/K2HPO4. Application of the magnetic field allowed improving the performance, with the best results being obtained at the longest distance between magnets (lowest magnetic field) and absence of NaCl (K = 0.93, PF = 4.22, Y = 83.79% and S = 0.09). The outcomes obtained demonstrate that ABS combination with low intensity magnetic field can be used as an efficient FFase pre-purification method.
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biochemical characterization and kinetic thermodynamic study of Aspergillus tamarii urm4634 β fructofuranosidase with transfructosylating activity
Biotechnology Progress, 2019Co-Authors: Rodrigo Lira De Oliveira, Attilio Converti, Marcos Fellipe Da Silva, Tatiana Souza PortoAbstract:This study reports on the biochemical characterization as well as the kinetic and thermodynamic study of Aspergillus tamarii URM4634 β-fructofuranosidase (FFase) with transfructosylating activity. Conditions for FFase activity were optimized by means of a central composite rotational design using pH and temperature as the independent variables, while residual activity tests carried out in the temperature range of 45-65°C enabled us to investigate FFase thermostability and estimate the kinetic and thermodynamic parameters of enzyme denaturation. Optimal conditions for sucrose hydrolysis and fructosyl transfer catalyzed by crude FFase were 50°C, and pH 6.0 and 7.4, respectively. The thermodynamic properties of irreversible enzyme inactivation were found to be activation energy of 293.1 kJ mol-1 , and activation enthalpy, entropy, and Gibbs free energy in the ranges 290.3-290.4 kJ mol-1 , 568.7-571.0 J mol-1 K-1 , and 97.9-108.8 kJ mol-1 , respectively. The results obtained in this study point out satisfactory enzyme activity and thermostability at temperatures commonly used for industrial fructo-oligosaccharide (FOS) synthesis; therefore, this novel FFase appears to be a promising biocatalyst with great potential for long-term FOS synthesis and invert sugar production. To the best of our knowledge, this is the first report on kinetic and thermodynamic parameters of an A. tamarii FFase.
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thermodynamic investigation of an alkaline protease from Aspergillus tamarii urm4634 a comparative approach between crude extract and purified enzyme
International Journal of Biological Macromolecules, 2017Co-Authors: Osmar Soares Da Silva, Rodrigo Lira De Oliveira, Jonatas De Carvalho Silva, Attilio Converti, Tatiana Souza PortoAbstract:Abstract The thermostable crude proteolytic extract and purified protease produced by Aspergillus tamarii URM4634 were investigated at different temperatures. The activity results were used to estimate the activation energy of the hydrolysis reaction catalyzed by crude extract and purified protease (E* = 34.2 and 16.2 kJ/mol) as well as the respective standard enthalpy variations of reversible enzyme unfolding (ΔH°u = 31.9 and 13.9 kJ/mol). When temperature was raised from 50 to 80 °C in residual activity tests, the specific rate constant of crude proteolytic extract thermoinactivation increased from 0.0072 to 0.0378 min−1, while that of purified protease from 0.0099 to 0.0235 min−1. These values, corresponding to half-life decreases from 96.3 to 18.3 min and from 70.0 to 29.5 min, respectively, enabled us to estimate the activation energy (E*d = 49.7 and 28.8 kJ/mol), enthalpy (ΔH*d = 47.0 and 26.1 kJ/mol), entropy (ΔS*d = −141.3 and −203.1 J/mol K) and Gibbs free energy (92.6 ≤ ΔG*d ≤ 96.6 kJ/mol and 91.8 ≤ ΔG*d ≤ 98.0 kJ/mol) of thermoinactivation. Such values suggest that this protease, which proved to be highly thermostable in both forms, could be profitably exploited in industrial applications. To the best of our knowledge, this is the first comparative study on thermodynamic parameters of a serine protease produced by Aspergillus tamarii URM4634.
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partitioning and extraction protease from Aspergillus tamarii urm4634 using peg citrate aqueous two phase systems
Biocatalysis and agricultural biotechnology, 2017Co-Authors: Osmar Soares Da Silva, Ana Lucia Figueiredo Porto, Rodrigo Lira De Oliveira, Attilio Converti, Matheus Henrique Gouveia Gomes, Tatiana Souza PortoAbstract:Abstract PEG-citrate Aqueous Two-Phase Systems (ATPS) were used to recover and partially purify protease from Aspergillus tamarii URM4634 produced by Solid State Fermentation. Experiments were performed according to a 2 4 -full factorial design using PEG molar mass ( M PEG ), PEG concentration ( C PEG ), citrate concentration ( C CIT ) and pH as independent variables; and purification factor ( PF ), partition coefficient ( K ) and activity yield ( Y ) as responses. Protease showed high activity in the PEG-rich phase, also M PEG and C CIT were shown to exert positive effects on all responses. The highest purification factor (3.95) was obtained using M PEG =8000 g/mol, 24% (w/w) C PEG , 20% (w/w) C CIT at pH 8.0. Consequently, the selected ATPS proved to be efficient and can be used as a first step for pre-purification of protease from solid state fermented of A. tamarii URM4634.
