Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal

Detalhes bibliográficos
Autor(a) principal: Monteiro, Paulo Sérgio
Data de Publicação: 2011
Tipo de documento: Tese
Idioma: por
Título da fonte: LOCUS Repositório Institucional da UFV
Texto Completo: http://locus.ufv.br/handle/123456789/305
Resumo: In this work, two fungi strains isolated from macadamia nut and sugar cane bagasse were evaluated for potential productions of phytases, focusing on biotechnological applications, mainly in animal feed. The fungi Rhizopus stolonifer isolated from macadamia nut was cultivated in submerged fermentation containing potato infusion, sucrose, peptone, Tween-20 and CaCl2, using response surface methodology (RSM) to optimize the phytase production. The use of MSR resulted in an enzyme activity of 0.65 U/mL, a value 9-times the initial phytase activity. The enzyme showed optimum pH and temperature in the range of 5.0-5.5 and of 65 oC, respectivamente, with t1/2 of 50 minutes at 50 oC. The KM app and Vmax app value were 0.692 mM and 0.780 μmol/min, respectively. The enzyme was efficient in phytate hydrolysis in animal feed, releasing 5.14 μmol PO4 3-/mL after 5 h of treatment. This value is equivalent to the initial phosphorus concentration. The fungi Aspergillus niger UFV-1 isolated from sugar cane bagasse was also cultivated in liquid medium containing potato infusion, sucrose, peptone, Tween-20 and CaCl2. After eight days of fermentation, the crude extract produced was collected and analyzed for enzymatic activity of phytases. The enzyme was purified by ultrafiltration, acid precipitation, and ion exchange and gel filtration chromatography. The phytase showed a molecular mass of 161 kDa determined by gel filtration and 81 kDa by SDS-PAGE electrophoresis, suggesting that the enzyme is composed of two subunits. The optimal values of pH and temperature were 2.0 and 60 oC, respectively. The kinetic parameters, KM and Vmax, determined by the Lineweaver-Burk plot, were 30.9 mM and 7.48 Tmol/min, respectively. The kcat estimated was 1.46 x 105 s-1 and kcat/KM was 4724919 s-1.M-1. The phytase showed high thermostability with t½ of 6.2 h at 80 oC and was resistant to pepsin and trypsin. Additionally, it efficiently in release of phosphorus, when evaluated in commercial animal feed, releasing 15.3 Tmol PO4 3-/mL, equivalent to 4-times the initial phosphorus concentration. The obtained results suggest that the two fungi strains evaluated have great potential as phytase producers for industrial application, mainly the strain Aspergillus niger UFV-1 that showed acidic optimum pH and high protease resistance.
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spelling Monteiro, Paulo Sérgiohttp://lattes.cnpq.br/9968171263899224Guimarães, Valéria Montezehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8Rezende, Sebastião Tavares dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3Lanna, Eduardo Arruda Teixeirahttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788116T3Oliveira, Eduardo Basílio dehttp://lattes.cnpq.br/4091528830821027Fontes, Elita Sciohttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794299J72015-03-26T12:15:16Z2012-07-062015-03-26T12:15:16Z2011-09-29MONTEIRO, Paulo Sérgio. Production and biochemical characterization of phytases from Rhizopus stolonifer and Aspergillus niger UFV-1 and its applications in animal feed. 2011. 79 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011.http://locus.ufv.br/handle/123456789/305In this work, two fungi strains isolated from macadamia nut and sugar cane bagasse were evaluated for potential productions of phytases, focusing on biotechnological applications, mainly in animal feed. The fungi Rhizopus stolonifer isolated from macadamia nut was cultivated in submerged fermentation containing potato infusion, sucrose, peptone, Tween-20 and CaCl2, using response surface methodology (RSM) to optimize the phytase production. The use of MSR resulted in an enzyme activity of 0.65 U/mL, a value 9-times the initial phytase activity. The enzyme showed optimum pH and temperature in the range of 5.0-5.5 and of 65 oC, respectivamente, with t1/2 of 50 minutes at 50 oC. The KM app and Vmax app value were 0.692 mM and 0.780 μmol/min, respectively. The enzyme was efficient in phytate hydrolysis in animal feed, releasing 5.14 μmol PO4 3-/mL after 5 h of treatment. This value is equivalent to the initial phosphorus concentration. The fungi Aspergillus niger UFV-1 isolated from sugar cane bagasse was also cultivated in liquid medium containing potato infusion, sucrose, peptone, Tween-20 and CaCl2. After eight days of fermentation, the crude extract produced was collected and analyzed for enzymatic activity of phytases. The enzyme was purified by ultrafiltration, acid precipitation, and ion exchange and gel filtration chromatography. The phytase showed a molecular mass of 161 kDa determined by gel filtration and 81 kDa by SDS-PAGE electrophoresis, suggesting that the enzyme is composed of two subunits. The optimal values of pH and temperature were 2.0 and 60 oC, respectively. The kinetic parameters, KM and Vmax, determined by the Lineweaver-Burk plot, were 30.9 mM and 7.48 Tmol/min, respectively. The kcat estimated was 1.46 x 105 s-1 and kcat/KM was 4724919 s-1.M-1. The phytase showed high thermostability with t½ of 6.2 h at 80 oC and was resistant to pepsin and trypsin. Additionally, it efficiently in release of phosphorus, when evaluated in commercial animal feed, releasing 15.3 Tmol PO4 3-/mL, equivalent to 4-times the initial phosphorus concentration. The obtained results suggest that the two fungi strains evaluated have great potential as phytase producers for industrial application, mainly the strain Aspergillus niger UFV-1 that showed acidic optimum pH and high protease resistance.Neste trabalho, duas cepas fúngicas isoladas da castanha macadâmia e de bagaço de cana-de-açúcar foram avaliadas quanto ao potencial para produção de fitases, visando aplicação em processos biotecnológicos, principalmente em rações animais. O fungo Rhizopus stolonifer, isolado a partir da macadâmia, foi cultivado em meio líquido contendo infusão de batata, sacarose, peptona, Tween-20, e CaCl2, utilizando a metodologia de superfície de resposta (MSR) para otimizar a produção de fitases. O uso da MSR resultou em uma atividade enzimática de 0,65 U/mL, valor nove vezes maior em relação à atividade inicial de fitases. A enzima apresentou pH e temperatura ótimas na faixa de 5,0-5,5 e 65 oC, respectivamente, com t1/2 de 50 minutos a 50 ºC. Os valores de KM app e Vmax app foram 0,692 mM e 0,780 Tmol/min, respectivamente. A enzima mostrou-se eficiente na hidrólise de fitato em ração animal, liberando 5,14 Tmol PO4 3-/mL após 5 horas de tratamento. Esse valor é equivalente à concentração inicial de fósforo. O fungo Aspergillus niger UFV-1, isolado de bagaço de cana-de-açúcar, também foi cultivada em meio líquido contendo infusão de batata, sacarose, peptona, Tween-20 e CaCl2. Após oito dias de incubação, o extrato enzimático produzido foi coletado e analisado para a atividade enzimática de fitases. A enzima foi purificada por ultrafiltração, precipitação ácida e cromatografias de troca iônica e gel filtração. A fitase apresentou massa molecular de 161 kDa, determinada por gel filtração e 81 kDa, determinada por eletroforese SDS-PAGE, sugerindo que a enzima é formada por duas subunidades. Os valores de pH e temperatura ótimos foram de 2,0 e 60 ºC, respectivamente. Os parâmetros cinéticos, KM e Vmax, determinados pela equação de Lineweaver-Burk, foram 30,9 mM e 7,48 Tmol/min, respectivamente. A kcat estimada foi de 1,46 x 105 s-1 e kcat/KM foi igual a 4724919 s-1.M-1. A fitase mostrou elevada termoestabilidade, com t1/2 de 6,2 horas a 80 ºC e foi resistente a pepsina e tripsina. Além disso, foi eficiente na liberação de fósforo, quando avaliada em ração animal comercial, liberando 15,3 Tmol PO4 3-/mL, valor equivalente a quatro vezes a concentração inicial de fósforo. Os resultados obtidos sugerem que as duas cepas fúngicas avaliadas têm grande potencial como produtoras de fitases para aplicação industrial, principalmente a cepa de Aspergillus niger UFV-1, cuja fitase apresentou pH ótimo ácido e elevada resistência a proteases.Fundação de Amparo a Pesquisa do Estado de Minas Geraisapplication/pdfporUniversidade Federal de ViçosaDoutorado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalFitasesFungosRação animalPhytasesFungiFeedCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIAProdução e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animalProduction and biochemical characterization of phytases from Rhizopus stolonifer and Aspergillus niger UFV-1 and its applications in animal feedinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf3113440https://locus.ufv.br//bitstream/123456789/305/1/texto%20completo.pdf32a7ebc272a0ea993c928efcf72d74c4MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain145900https://locus.ufv.br//bitstream/123456789/305/2/texto%20completo.pdf.txt75089b6fe127a91e267bdc4ea594fb33MD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3579https://locus.ufv.br//bitstream/123456789/305/3/texto%20completo.pdf.jpgdcbe979024399313b27e22f1276ee321MD53123456789/3052016-04-06 23:01:37.493oai:locus.ufv.br:123456789/305Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452016-04-07T02:01:37LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false
dc.title.por.fl_str_mv Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
dc.title.alternative.eng.fl_str_mv Production and biochemical characterization of phytases from Rhizopus stolonifer and Aspergillus niger UFV-1 and its applications in animal feed
title Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
spellingShingle Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
Monteiro, Paulo Sérgio
Fitases
Fungos
Ração animal
Phytases
Fungi
Feed
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
title_short Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
title_full Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
title_fullStr Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
title_full_unstemmed Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
title_sort Produção e caracterização bioquímica de fitases de Rhizopus stolonifer e Aspergillus niger UFV-1 e suas aplicações em ração animal
author Monteiro, Paulo Sérgio
author_facet Monteiro, Paulo Sérgio
author_role author
dc.contributor.authorLattes.por.fl_str_mv http://lattes.cnpq.br/9968171263899224
dc.contributor.author.fl_str_mv Monteiro, Paulo Sérgio
dc.contributor.advisor-co1.fl_str_mv Guimarães, Valéria Monteze
dc.contributor.advisor-co1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3
dc.contributor.advisor-co2.fl_str_mv Fietto, Luciano Gomes
dc.contributor.advisor-co2Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8
dc.contributor.advisor1.fl_str_mv Rezende, Sebastião Tavares de
dc.contributor.advisor1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3
dc.contributor.referee1.fl_str_mv Lanna, Eduardo Arruda Teixeira
dc.contributor.referee1Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4788116T3
dc.contributor.referee2.fl_str_mv Oliveira, Eduardo Basílio de
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/4091528830821027
dc.contributor.referee3.fl_str_mv Fontes, Elita Scio
dc.contributor.referee3Lattes.fl_str_mv http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4794299J7
contributor_str_mv Guimarães, Valéria Monteze
Fietto, Luciano Gomes
Rezende, Sebastião Tavares de
Lanna, Eduardo Arruda Teixeira
Oliveira, Eduardo Basílio de
Fontes, Elita Scio
dc.subject.por.fl_str_mv Fitases
Fungos
Ração animal
topic Fitases
Fungos
Ração animal
Phytases
Fungi
Feed
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
dc.subject.eng.fl_str_mv Phytases
Fungi
Feed
dc.subject.cnpq.fl_str_mv CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA::ENZIMOLOGIA
description In this work, two fungi strains isolated from macadamia nut and sugar cane bagasse were evaluated for potential productions of phytases, focusing on biotechnological applications, mainly in animal feed. The fungi Rhizopus stolonifer isolated from macadamia nut was cultivated in submerged fermentation containing potato infusion, sucrose, peptone, Tween-20 and CaCl2, using response surface methodology (RSM) to optimize the phytase production. The use of MSR resulted in an enzyme activity of 0.65 U/mL, a value 9-times the initial phytase activity. The enzyme showed optimum pH and temperature in the range of 5.0-5.5 and of 65 oC, respectivamente, with t1/2 of 50 minutes at 50 oC. The KM app and Vmax app value were 0.692 mM and 0.780 μmol/min, respectively. The enzyme was efficient in phytate hydrolysis in animal feed, releasing 5.14 μmol PO4 3-/mL after 5 h of treatment. This value is equivalent to the initial phosphorus concentration. The fungi Aspergillus niger UFV-1 isolated from sugar cane bagasse was also cultivated in liquid medium containing potato infusion, sucrose, peptone, Tween-20 and CaCl2. After eight days of fermentation, the crude extract produced was collected and analyzed for enzymatic activity of phytases. The enzyme was purified by ultrafiltration, acid precipitation, and ion exchange and gel filtration chromatography. The phytase showed a molecular mass of 161 kDa determined by gel filtration and 81 kDa by SDS-PAGE electrophoresis, suggesting that the enzyme is composed of two subunits. The optimal values of pH and temperature were 2.0 and 60 oC, respectively. The kinetic parameters, KM and Vmax, determined by the Lineweaver-Burk plot, were 30.9 mM and 7.48 Tmol/min, respectively. The kcat estimated was 1.46 x 105 s-1 and kcat/KM was 4724919 s-1.M-1. The phytase showed high thermostability with t½ of 6.2 h at 80 oC and was resistant to pepsin and trypsin. Additionally, it efficiently in release of phosphorus, when evaluated in commercial animal feed, releasing 15.3 Tmol PO4 3-/mL, equivalent to 4-times the initial phosphorus concentration. The obtained results suggest that the two fungi strains evaluated have great potential as phytase producers for industrial application, mainly the strain Aspergillus niger UFV-1 that showed acidic optimum pH and high protease resistance.
publishDate 2011
dc.date.issued.fl_str_mv 2011-09-29
dc.date.available.fl_str_mv 2012-07-06
2015-03-26T12:15:16Z
dc.date.accessioned.fl_str_mv 2015-03-26T12:15:16Z
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dc.type.driver.fl_str_mv info:eu-repo/semantics/doctoralThesis
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dc.identifier.citation.fl_str_mv MONTEIRO, Paulo Sérgio. Production and biochemical characterization of phytases from Rhizopus stolonifer and Aspergillus niger UFV-1 and its applications in animal feed. 2011. 79 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011.
dc.identifier.uri.fl_str_mv http://locus.ufv.br/handle/123456789/305
identifier_str_mv MONTEIRO, Paulo Sérgio. Production and biochemical characterization of phytases from Rhizopus stolonifer and Aspergillus niger UFV-1 and its applications in animal feed. 2011. 79 f. Tese (Doutorado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2011.
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dc.publisher.none.fl_str_mv Universidade Federal de Viçosa
dc.publisher.program.fl_str_mv Doutorado em Bioquímica Agrícola
dc.publisher.initials.fl_str_mv UFV
dc.publisher.country.fl_str_mv BR
dc.publisher.department.fl_str_mv Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal
publisher.none.fl_str_mv Universidade Federal de Viçosa
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