β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar
Autor(a) principal: | |
---|---|
Data de Publicação: | 2014 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | LOCUS Repositório Institucional da UFV |
Texto Completo: | http://locus.ufv.br/handle/123456789/2475 |
Resumo: | In this work, a β-glucosidases complex from the phytopathogen fungus Chrysoporthe cubensis LPF-1 was purified, biochemistry and kinetically characterized and evaluated for its potential application in the process of saccharification of sugarcane bagasse. C. cubensis was grown in solid state fermentation (SSF) contaning wheat bran as carbon source and after five days of fermentation the enzymatic extract was obtaneid. A Ec complex containing three β-glucosidases was purified by ion exchange and gel filtration chromatographies and the end of the process the Ec showed a purification factor of 2.05 times with a yield of 64.5%. The molecular weight of the β-glucosidases was as 85,43 and 74,17 kDa for E2 and E3, respectively, and for E1a, E1b, E1c was 63,09, 60,25 e 56,23 kDa, respectively, when estimated by SDS-PAGE. The complex of β-glucosidases (Ec) exhibited maximum activity at pH 4.0 and 60 ° C. Ec was stable in the pH range of 2.0- 8.0 and showed high thermal stability at temperatures of 50, 60 and 70 ° C. The Ec half- life values were 52 hours and 39 minutes, 35 hours and 30 minutes and 15.3 minutes at 50, 60 and 70 ° C, respectively. The Ec of β-glucosidase activity was completely innibited by HgCl2 and MnSO4 at a concentration of 10 mM and the complex of β- glucosidases was specific for glucose residues in β position. The KM values for Ec of 0.132 and 0.7816 mM and Vmax of 0.416 and 0.3414 μM.min-1 were obtained using the ρNPβGlc and cellobiose substrates, respectively. The Ec showed competitive inhibition of glucose to type with Ki value of 8.43 mM. The β-glucosidases were employed in saccharification of sugarcane bagasse, previously submitted to the alkaline pretreatment. Ec was used as a source of supplementation for both extract from C. cubensis as well as in a commercial cellulase cocktail. The saccharification results with the supplemented preparations with β-glucosidases were compared with those obtained employing a commercial cocktail and only the extract of C. cubensis and the production of glucose and xylose were evaluated for 72 hours of hydrolysis. Saccharification performed with commercial cocktail supplemented with the β-glucosidases promoted the release of 2.82 g / L of glucose and 0.98 g / L xylose whereas only with the commercial cocktail were released 1.78 g / L of glucose and 0.85 g / L of xylose. On the other hand, the saccharification with the C. cubensis extract produced 1.69 g / L of glucose and 1.64 g / L xylose while the same fraction supplemented with β-glucosidases promoted the release of 2.62 g / L of glucose and 2.65 g / L of xylose. Thus, the results obtained suggests that the β-glucosidases of C. cubensis evaluated in this work have potential to be used as a source of supplemental other enzyme preparations, since the hydrolysis efficiency was higher than that observed in commercial cocktail of cellulase as well as extract of C. cubensis. |
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Andrade, Lorena Gusmão Alvarenga dehttp://lattes.cnpq.br/5678203179976910Guimarães, Valéria Montezehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3Fietto, Luciano Gomeshttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8Silveira, Wendel Batista dahttp://lattes.cnpq.br/7361036485940798Rezende, Sebastião Tavares dehttp://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A32015-03-26T13:07:39Z2015-01-202015-03-26T13:07:39Z2014-08-14ANDRADE, Lorena Gusmão Alvarenga de. β-Glucosidase of the phytopathogen fungus Chrysoporthe cubensis LPF-1: purification, characterization and application of sugarcane bagasse saccharification. 2014. 70 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2014.http://locus.ufv.br/handle/123456789/2475In this work, a β-glucosidases complex from the phytopathogen fungus Chrysoporthe cubensis LPF-1 was purified, biochemistry and kinetically characterized and evaluated for its potential application in the process of saccharification of sugarcane bagasse. C. cubensis was grown in solid state fermentation (SSF) contaning wheat bran as carbon source and after five days of fermentation the enzymatic extract was obtaneid. A Ec complex containing three β-glucosidases was purified by ion exchange and gel filtration chromatographies and the end of the process the Ec showed a purification factor of 2.05 times with a yield of 64.5%. The molecular weight of the β-glucosidases was as 85,43 and 74,17 kDa for E2 and E3, respectively, and for E1a, E1b, E1c was 63,09, 60,25 e 56,23 kDa, respectively, when estimated by SDS-PAGE. The complex of β-glucosidases (Ec) exhibited maximum activity at pH 4.0 and 60 ° C. Ec was stable in the pH range of 2.0- 8.0 and showed high thermal stability at temperatures of 50, 60 and 70 ° C. The Ec half- life values were 52 hours and 39 minutes, 35 hours and 30 minutes and 15.3 minutes at 50, 60 and 70 ° C, respectively. The Ec of β-glucosidase activity was completely innibited by HgCl2 and MnSO4 at a concentration of 10 mM and the complex of β- glucosidases was specific for glucose residues in β position. The KM values for Ec of 0.132 and 0.7816 mM and Vmax of 0.416 and 0.3414 μM.min-1 were obtained using the ρNPβGlc and cellobiose substrates, respectively. The Ec showed competitive inhibition of glucose to type with Ki value of 8.43 mM. The β-glucosidases were employed in saccharification of sugarcane bagasse, previously submitted to the alkaline pretreatment. Ec was used as a source of supplementation for both extract from C. cubensis as well as in a commercial cellulase cocktail. The saccharification results with the supplemented preparations with β-glucosidases were compared with those obtained employing a commercial cocktail and only the extract of C. cubensis and the production of glucose and xylose were evaluated for 72 hours of hydrolysis. Saccharification performed with commercial cocktail supplemented with the β-glucosidases promoted the release of 2.82 g / L of glucose and 0.98 g / L xylose whereas only with the commercial cocktail were released 1.78 g / L of glucose and 0.85 g / L of xylose. On the other hand, the saccharification with the C. cubensis extract produced 1.69 g / L of glucose and 1.64 g / L xylose while the same fraction supplemented with β-glucosidases promoted the release of 2.62 g / L of glucose and 2.65 g / L of xylose. Thus, the results obtained suggests that the β-glucosidases of C. cubensis evaluated in this work have potential to be used as a source of supplemental other enzyme preparations, since the hydrolysis efficiency was higher than that observed in commercial cocktail of cellulase as well as extract of C. cubensis.Neste trabalho, um complexo de β-glicosidases do fungo fitopatógeno Chrysoporthe cubensis LPF-1 foi purificado, caracterizado bioquímica e cineticamente e avaliado quanto ao seu potencial em aplicação no processo de sacarificação de bagaço de cana de açúcar. O fungo C. cubensis foi cultivado sob fermentação no estado sólido (SSF) contendo farelo de trigo como fonte de carbono e, após 5 dias de fermentação o extrato enzimático foi obtido. O conjunto de três β-glicosidases, foi purificado por cromatografias de troca iônica e filtração em gel e, ao final do processo, apresentou um fator de purificação de 2,05 vezes com um rendimento de 64,5 %. As massas moleculares das β-glicosidases foram de 85,43 e 74,17 kDa, para E2 e E3, respectivamente, e de 63,09, 60,25 e 56,23 kDa para E1a, E1b e E1c, respectivamente, quando estimadas por SDS-PAGE. O complexo de β-glicosidases (Ec) exibiu atividade máxima em pH 4,0 e na temperatura de 60°C. Ec foi estável na faixa de pH de 2,0-8,0 e apresentou alta termoestabilidade nas temperaturas de 50, 60 e 70 °C. Os valores de meia-vida para Ec foram de 52 horas e 39 minutos, 35 horas e 30 minutos e, 15,3 minutos, a 50, 60 e 70 °C, respectivamente. A atividade de β-glicosidase de Ec foi completamente inibida por HgCl2 e MnSO4, na concentração de 10 mM e, o complexo de β-glicosidases apresentou especificidade absoluta para resíduos de glicose com ligações do tipo β. Os valores de KM para Ec de 0,132 mM e 0,7816 mM e Vmáx de 0,416 μM.min-1 e Vmáx 0,3414 μM.min-1, foram obtidos utilizando os substratos ρNPβGlc e celobiose, respectivamente. Ec apresentou inibição do tipo competitiva para a glicose com valor de Ki 8,43 mM. As β-glicosidases foram utilizadas em experimentos de sacarificação do bagaço de cana, submetido previamente ao pré- tratamento alcalino. Ec foi utilizado para suplementação tanto do extrato proveniente do C. cubensis como também para um coquetel comercial de celulases. Os resultados da sacarificação com as preparações suplementadas com as β-glicosidases foram comparados com aqueles obtidos utilizando somente o coquetel comercial e o extrato do C. cubensis e, a produção de glicose e xilose foi avaliada durante 72 horas de hidrólise. A sacarificação realizada com o coquetel comercial suplementado com as β-glicosidases promoveu a liberação de 2,82 g/L de glicose e 0,98 g/L de xilose ao passo que, somente com o coquetel comercial foram liberados 1,78 g/L de glicose e 0,85 g/L de xilose. Por outro lado, a sacarificação com o extrato do C. cubensis liberou 1,69 g/L de glicose e 1,64 g/L de xilose enquanto, que o mesmo extrato suplementado com as β-glicosidases, promoveu a liberação de 2,62 g/L de glicose e 2,65 g/L de xilose. Dessa forma, estes resultados demonstram que as β-glicosidases do C. cubensis apresentam potencial para serem utilizadas como fonte de suplementação em outras preparações enzimáticas, visto que a eficiência de hidrólise observada foi superior à observada com o coquetel comercial de celulases bem como com o extrato de C. cubensis.Conselho Nacional de Desenvolvimento Científico e Tecnológicoapplication/pdfporUniversidade Federal de ViçosaMestrado em Bioquímica AgrícolaUFVBRBioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animalFungoChrysoporthe cubensisβ-GlicosidasesBagaço de canaPurificaçãoFungusChrysoporthe cubensisβ-glucosidasesBagassePurificationCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAβ-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcarβ-Glucosidase of the phytopathogen fungus Chrysoporthe cubensis LPF-1: purification, characterization and application of sugarcane bagasse saccharificationinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:LOCUS Repositório Institucional da UFVinstname:Universidade Federal de Viçosa (UFV)instacron:UFVORIGINALtexto completo.pdfapplication/pdf808595https://locus.ufv.br//bitstream/123456789/2475/1/texto%20completo.pdf518b39ebd08965bbb817f6e841cc2c05MD51TEXTtexto completo.pdf.txttexto completo.pdf.txtExtracted texttext/plain119131https://locus.ufv.br//bitstream/123456789/2475/2/texto%20completo.pdf.txt07d1cf815fdca0d576a556705e0802adMD52THUMBNAILtexto completo.pdf.jpgtexto completo.pdf.jpgIM Thumbnailimage/jpeg3768https://locus.ufv.br//bitstream/123456789/2475/3/texto%20completo.pdf.jpg343ba040e1ba9f32f1ab6e12ffb6e451MD53123456789/24752017-10-06 15:28:19.402oai:locus.ufv.br:123456789/2475Repositório InstitucionalPUBhttps://www.locus.ufv.br/oai/requestfabiojreis@ufv.bropendoar:21452017-10-06T18:28:19LOCUS Repositório Institucional da UFV - Universidade Federal de Viçosa (UFV)false |
dc.title.por.fl_str_mv |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
dc.title.alternative.eng.fl_str_mv |
β-Glucosidase of the phytopathogen fungus Chrysoporthe cubensis LPF-1: purification, characterization and application of sugarcane bagasse saccharification |
title |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
spellingShingle |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar Andrade, Lorena Gusmão Alvarenga de Fungo Chrysoporthe cubensis β-Glicosidases Bagaço de cana Purificação Fungus Chrysoporthe cubensis β-glucosidases Bagasse Purification CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
title_short |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
title_full |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
title_fullStr |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
title_full_unstemmed |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
title_sort |
β-Glicosidases do fungo fitopatógeno Chrysoporthe cubensis: purificação, caracterização e aplicação na sacarificação do bagaço de cana-de- açúcar |
author |
Andrade, Lorena Gusmão Alvarenga de |
author_facet |
Andrade, Lorena Gusmão Alvarenga de |
author_role |
author |
dc.contributor.authorLattes.por.fl_str_mv |
http://lattes.cnpq.br/5678203179976910 |
dc.contributor.author.fl_str_mv |
Andrade, Lorena Gusmão Alvarenga de |
dc.contributor.advisor1.fl_str_mv |
Guimarães, Valéria Monteze |
dc.contributor.advisor1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4798758T3 |
dc.contributor.referee1.fl_str_mv |
Fietto, Luciano Gomes |
dc.contributor.referee1Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4763824H8 |
dc.contributor.referee2.fl_str_mv |
Silveira, Wendel Batista da |
dc.contributor.referee2Lattes.fl_str_mv |
http://lattes.cnpq.br/7361036485940798 |
dc.contributor.referee3.fl_str_mv |
Rezende, Sebastião Tavares de |
dc.contributor.referee3Lattes.fl_str_mv |
http://buscatextual.cnpq.br/buscatextual/visualizacv.do?id=K4787599A3 |
contributor_str_mv |
Guimarães, Valéria Monteze Fietto, Luciano Gomes Silveira, Wendel Batista da Rezende, Sebastião Tavares de |
dc.subject.por.fl_str_mv |
Fungo Chrysoporthe cubensis β-Glicosidases Bagaço de cana Purificação |
topic |
Fungo Chrysoporthe cubensis β-Glicosidases Bagaço de cana Purificação Fungus Chrysoporthe cubensis β-glucosidases Bagasse Purification CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.subject.eng.fl_str_mv |
Fungus Chrysoporthe cubensis β-glucosidases Bagasse Purification |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
description |
In this work, a β-glucosidases complex from the phytopathogen fungus Chrysoporthe cubensis LPF-1 was purified, biochemistry and kinetically characterized and evaluated for its potential application in the process of saccharification of sugarcane bagasse. C. cubensis was grown in solid state fermentation (SSF) contaning wheat bran as carbon source and after five days of fermentation the enzymatic extract was obtaneid. A Ec complex containing three β-glucosidases was purified by ion exchange and gel filtration chromatographies and the end of the process the Ec showed a purification factor of 2.05 times with a yield of 64.5%. The molecular weight of the β-glucosidases was as 85,43 and 74,17 kDa for E2 and E3, respectively, and for E1a, E1b, E1c was 63,09, 60,25 e 56,23 kDa, respectively, when estimated by SDS-PAGE. The complex of β-glucosidases (Ec) exhibited maximum activity at pH 4.0 and 60 ° C. Ec was stable in the pH range of 2.0- 8.0 and showed high thermal stability at temperatures of 50, 60 and 70 ° C. The Ec half- life values were 52 hours and 39 minutes, 35 hours and 30 minutes and 15.3 minutes at 50, 60 and 70 ° C, respectively. The Ec of β-glucosidase activity was completely innibited by HgCl2 and MnSO4 at a concentration of 10 mM and the complex of β- glucosidases was specific for glucose residues in β position. The KM values for Ec of 0.132 and 0.7816 mM and Vmax of 0.416 and 0.3414 μM.min-1 were obtained using the ρNPβGlc and cellobiose substrates, respectively. The Ec showed competitive inhibition of glucose to type with Ki value of 8.43 mM. The β-glucosidases were employed in saccharification of sugarcane bagasse, previously submitted to the alkaline pretreatment. Ec was used as a source of supplementation for both extract from C. cubensis as well as in a commercial cellulase cocktail. The saccharification results with the supplemented preparations with β-glucosidases were compared with those obtained employing a commercial cocktail and only the extract of C. cubensis and the production of glucose and xylose were evaluated for 72 hours of hydrolysis. Saccharification performed with commercial cocktail supplemented with the β-glucosidases promoted the release of 2.82 g / L of glucose and 0.98 g / L xylose whereas only with the commercial cocktail were released 1.78 g / L of glucose and 0.85 g / L of xylose. On the other hand, the saccharification with the C. cubensis extract produced 1.69 g / L of glucose and 1.64 g / L xylose while the same fraction supplemented with β-glucosidases promoted the release of 2.62 g / L of glucose and 2.65 g / L of xylose. Thus, the results obtained suggests that the β-glucosidases of C. cubensis evaluated in this work have potential to be used as a source of supplemental other enzyme preparations, since the hydrolysis efficiency was higher than that observed in commercial cocktail of cellulase as well as extract of C. cubensis. |
publishDate |
2014 |
dc.date.issued.fl_str_mv |
2014-08-14 |
dc.date.accessioned.fl_str_mv |
2015-03-26T13:07:39Z |
dc.date.available.fl_str_mv |
2015-01-20 2015-03-26T13:07:39Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
format |
masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
ANDRADE, Lorena Gusmão Alvarenga de. β-Glucosidase of the phytopathogen fungus Chrysoporthe cubensis LPF-1: purification, characterization and application of sugarcane bagasse saccharification. 2014. 70 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2014. |
dc.identifier.uri.fl_str_mv |
http://locus.ufv.br/handle/123456789/2475 |
identifier_str_mv |
ANDRADE, Lorena Gusmão Alvarenga de. β-Glucosidase of the phytopathogen fungus Chrysoporthe cubensis LPF-1: purification, characterization and application of sugarcane bagasse saccharification. 2014. 70 f. Dissertação (Mestrado em Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal) - Universidade Federal de Viçosa, Viçosa, 2014. |
url |
http://locus.ufv.br/handle/123456789/2475 |
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por |
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por |
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Universidade Federal de Viçosa |
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Mestrado em Bioquímica Agrícola |
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UFV |
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BR |
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Bioquímica e Biologia molecular de plantas; Bioquímica e Biologia molecular animal |
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Universidade Federal de Viçosa |
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