Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres
Autor(a) principal: | |
---|---|
Data de Publicação: | 2012 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/4099 |
Resumo: | Lipases (triacyl-glycerol-hydrolases) are enzymes that catalyze hydrolysis, esterification and transesterification reactions. Lipase can be obtained from animals, microbial and vegetable sources. Nowadays, commercial lipases are majority produced from microbial sources. The use of these enzymes in industrial scale is still limited because of its high cost of production, favoring then, the search for new sources of lipases. This work aimed the utilization of oilseeds as lipase sources, aiming its use in the synthesis of fatty esters and in the hydrolysis of vegetable oils. To achieve this goal, the protein content of seeds of sunflower, castor bean and soybean was solubilized in buffered medium. The oilseeds were crushed in the presence of sodium phosphate buffer pH 7.0 (50 mM), followed by 11 hconstant stirring at room temperature. Under these conditions, the average productivities were ca. 237, 100 and 81 U/g of dried seeds. The solids were withdrawal from the crude extract by filtration, followed by centrifugation. The clarified crude extract was purified by ultrafiltration in 100 kDa cut-off polypropylene membrane. This procedure allows an activity recovery of 40, 35 and 11% for soybean, sunflower and castor bean, respectively. The purified lipases from soybean, sunflower and castor bean seeds were immobilized on hydrophobic support (silica-octyl) by interfacial adsorption, yielding biocatalysts with recovered activities of 683%, 413% and 1494%, respectively. SDS-PAGE electrophoresis and activity assays during the immobilization of the purified lipases on silica-octil suggested the presence of two lipase isoforms with molecular weights around of 20 and 30 kDa. Soluble soybean lipase exhibited optimum pH and temperature for hydrolysis of olive oil around 8.0 and 47 °C, respectively, while for immobilized soybean lipase (derivative) were 6.0 and 57°C, respectively. The halflife of the immobilized lipase at 50oC and pH 7 was around 8 h. The synthesis of butyl butyrate at 40oC catalyzed by immobilized lipase yield a conversion of approximately 15% after 9 h of reaction. The productivity of lipases from soybean seeds can be increased by germination of the seeds for 12 h, followed by extraction at 25oC for 12 h with salt solution (sodium phosphate buffer pH 7.0) at 100 mM concentration, supplemented with 1% (m/v) Tris-HCl. |
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Vescovi, ViniciusTardioli, Paulo Waldirhttp://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4790436Z8http://lattes.cnpq.br/758873384295891966dd02a5-d9bb-4e6c-9484-4bb2a30093862016-06-02T19:56:48Z2012-10-032016-06-02T19:56:48Z2012-03-30VESCOVI, Vinicius. Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres. 2012. 93 f. Dissertação (Mestrado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2012.https://repositorio.ufscar.br/handle/ufscar/4099Lipases (triacyl-glycerol-hydrolases) are enzymes that catalyze hydrolysis, esterification and transesterification reactions. Lipase can be obtained from animals, microbial and vegetable sources. Nowadays, commercial lipases are majority produced from microbial sources. The use of these enzymes in industrial scale is still limited because of its high cost of production, favoring then, the search for new sources of lipases. This work aimed the utilization of oilseeds as lipase sources, aiming its use in the synthesis of fatty esters and in the hydrolysis of vegetable oils. To achieve this goal, the protein content of seeds of sunflower, castor bean and soybean was solubilized in buffered medium. The oilseeds were crushed in the presence of sodium phosphate buffer pH 7.0 (50 mM), followed by 11 hconstant stirring at room temperature. Under these conditions, the average productivities were ca. 237, 100 and 81 U/g of dried seeds. The solids were withdrawal from the crude extract by filtration, followed by centrifugation. The clarified crude extract was purified by ultrafiltration in 100 kDa cut-off polypropylene membrane. This procedure allows an activity recovery of 40, 35 and 11% for soybean, sunflower and castor bean, respectively. The purified lipases from soybean, sunflower and castor bean seeds were immobilized on hydrophobic support (silica-octyl) by interfacial adsorption, yielding biocatalysts with recovered activities of 683%, 413% and 1494%, respectively. SDS-PAGE electrophoresis and activity assays during the immobilization of the purified lipases on silica-octil suggested the presence of two lipase isoforms with molecular weights around of 20 and 30 kDa. Soluble soybean lipase exhibited optimum pH and temperature for hydrolysis of olive oil around 8.0 and 47 °C, respectively, while for immobilized soybean lipase (derivative) were 6.0 and 57°C, respectively. The halflife of the immobilized lipase at 50oC and pH 7 was around 8 h. The synthesis of butyl butyrate at 40oC catalyzed by immobilized lipase yield a conversion of approximately 15% after 9 h of reaction. The productivity of lipases from soybean seeds can be increased by germination of the seeds for 12 h, followed by extraction at 25oC for 12 h with salt solution (sodium phosphate buffer pH 7.0) at 100 mM concentration, supplemented with 1% (m/v) Tris-HCl.Lipases (glicerol éster hidrolases, EC 3.1.1.3) catalisam reações de hidrólise, esterificação e transesterificação. As lipases podem ser obtidas de fontes animais, microbianas e vegetais, sendo que as de origem microbiana representam a grande maioria das lipases produzidas atualmente. No entanto, o uso dessas enzimas em escala industrial ainda é restrito devido ao alto custo de produção, favorecendo, assim, a busca por novas fontes de lipase. Este trabalho teve por objetivo a utilização de sementes de oleaginosas como fontes de lipases, visando sua aplicação na síntese de ésteres de ácidos graxos e hidrólise de óleos vegetais. Inicialmente sementes de soja, girassol e mamona foram trituradas em tampão fosfato de sódio 50 mM, pH 7, seguida por agitação de 11 horas em temperatura ambiente. Sob essas condições, as produtividades médias foram de aproximadamente 237, 100 e 81 U/g de sementes secas. Os sólidos foram removidos do extrato enzimático bruto por filtração e o extrato enzimático foi clarificado por centrifugação. O extrato clarificado foi purificado por ultrafiltração em membrana de polipropileno com diâmetro de corte de 100 kDa. Esse procedimento permitiu a recuperação de 40, 35 e 11% da atividade inicialmente presente nos extratos enzimáticos brutos obtidos a partir de sementes de soja, girassol e mamona, respectivamente. Lipases de sementes de soja, girassol e mamona foram imobilizadas por adsorção hidrofóbica em sílica ativada com grupos octil (sílica-octil), obtendo-se biocatalisadores com atividades recuperadas de 683%, 413% e 1494%, respectivamente. Eletroforese SDS-PAGE do extrato enzimático da soja e ensaios de atividade durante a imobilização em sílica-octil sugeriram a presença de duas isoformas de lipases, com massas moleculares de aproximadamente 20 e 30 kDa. O pH e a temperatura de máximas atividades hidrolíticas do extrato enzimático da soja foram de 8,0 e 47ºC, respectivamente, enquanto para a enzima imobilizada foram de 6,0 e 57ºC, respectivamente. O tempo de meia-vida da enzima imobilizada a 50ºC e pH 7 foi de 8 h. Na síntese de butirato de butila, realizada a 40ºC, obteve-se uma conversão de aproximadamente 15% em 9 h de reação. A produtividade de lipases de sementes de soja pode ser aumentada por germinação das sementes por 12 h, seguida da extração a 25ºC por 12 h com solução salina (tampão fosfato de sódio, pH 7,0) com uma concentração de 100 mM e adição de Tris-HCl 1% (m/v).Agência Nacional de Petróleoapplication/pdfporUniversidade Federal de São CarlosPrograma de Pós-Graduação em Engenharia Química - PPGEQUFSCarBREngenharia químicaExtraçãoEnzimas - purificaçãoLipaseImobilizaçãoAdsorção hidrofóbicaSílica-octilExtractionPurificationPlant lipaseImmobilizationHydrophobic adsorptionSilica-octylENGENHARIAS::ENGENHARIA QUIMICAExtração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteresinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesis-1-157a91b28-06b2-4fc7-b127-2a5005569c49info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL4556.pdfapplication/pdf4130912https://repositorio.ufscar.br/bitstream/ufscar/4099/1/4556.pdfce52d6d080c273997017ee16e040fc29MD51TEXT4556.pdf.txt4556.pdf.txtExtracted texttext/plain0https://repositorio.ufscar.br/bitstream/ufscar/4099/2/4556.pdf.txtd41d8cd98f00b204e9800998ecf8427eMD52THUMBNAIL4556.pdf.jpg4556.pdf.jpgIM Thumbnailimage/jpeg7475https://repositorio.ufscar.br/bitstream/ufscar/4099/3/4556.pdf.jpg66e8cb088065c14cad00aac1814188ccMD53ufscar/40992023-09-18 18:31:33.597oai:repositorio.ufscar.br:ufscar/4099Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:33Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
title |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
spellingShingle |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres Vescovi, Vinicius Engenharia química Extração Enzimas - purificação Lipase Imobilização Adsorção hidrofóbica Sílica-octil Extraction Purification Plant lipase Immobilization Hydrophobic adsorption Silica-octyl ENGENHARIAS::ENGENHARIA QUIMICA |
title_short |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
title_full |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
title_fullStr |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
title_full_unstemmed |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
title_sort |
Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres |
author |
Vescovi, Vinicius |
author_facet |
Vescovi, Vinicius |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/7588733842958919 |
dc.contributor.author.fl_str_mv |
Vescovi, Vinicius |
dc.contributor.advisor1.fl_str_mv |
Tardioli, Paulo Waldir |
dc.contributor.advisor1Lattes.fl_str_mv |
http://genos.cnpq.br:12010/dwlattes/owa/prc_imp_cv_int?f_cod=K4790436Z8 |
dc.contributor.authorID.fl_str_mv |
66dd02a5-d9bb-4e6c-9484-4bb2a3009386 |
contributor_str_mv |
Tardioli, Paulo Waldir |
dc.subject.por.fl_str_mv |
Engenharia química Extração Enzimas - purificação Lipase Imobilização Adsorção hidrofóbica Sílica-octil |
topic |
Engenharia química Extração Enzimas - purificação Lipase Imobilização Adsorção hidrofóbica Sílica-octil Extraction Purification Plant lipase Immobilization Hydrophobic adsorption Silica-octyl ENGENHARIAS::ENGENHARIA QUIMICA |
dc.subject.eng.fl_str_mv |
Extraction Purification Plant lipase Immobilization Hydrophobic adsorption Silica-octyl |
dc.subject.cnpq.fl_str_mv |
ENGENHARIAS::ENGENHARIA QUIMICA |
description |
Lipases (triacyl-glycerol-hydrolases) are enzymes that catalyze hydrolysis, esterification and transesterification reactions. Lipase can be obtained from animals, microbial and vegetable sources. Nowadays, commercial lipases are majority produced from microbial sources. The use of these enzymes in industrial scale is still limited because of its high cost of production, favoring then, the search for new sources of lipases. This work aimed the utilization of oilseeds as lipase sources, aiming its use in the synthesis of fatty esters and in the hydrolysis of vegetable oils. To achieve this goal, the protein content of seeds of sunflower, castor bean and soybean was solubilized in buffered medium. The oilseeds were crushed in the presence of sodium phosphate buffer pH 7.0 (50 mM), followed by 11 hconstant stirring at room temperature. Under these conditions, the average productivities were ca. 237, 100 and 81 U/g of dried seeds. The solids were withdrawal from the crude extract by filtration, followed by centrifugation. The clarified crude extract was purified by ultrafiltration in 100 kDa cut-off polypropylene membrane. This procedure allows an activity recovery of 40, 35 and 11% for soybean, sunflower and castor bean, respectively. The purified lipases from soybean, sunflower and castor bean seeds were immobilized on hydrophobic support (silica-octyl) by interfacial adsorption, yielding biocatalysts with recovered activities of 683%, 413% and 1494%, respectively. SDS-PAGE electrophoresis and activity assays during the immobilization of the purified lipases on silica-octil suggested the presence of two lipase isoforms with molecular weights around of 20 and 30 kDa. Soluble soybean lipase exhibited optimum pH and temperature for hydrolysis of olive oil around 8.0 and 47 °C, respectively, while for immobilized soybean lipase (derivative) were 6.0 and 57°C, respectively. The halflife of the immobilized lipase at 50oC and pH 7 was around 8 h. The synthesis of butyl butyrate at 40oC catalyzed by immobilized lipase yield a conversion of approximately 15% after 9 h of reaction. The productivity of lipases from soybean seeds can be increased by germination of the seeds for 12 h, followed by extraction at 25oC for 12 h with salt solution (sodium phosphate buffer pH 7.0) at 100 mM concentration, supplemented with 1% (m/v) Tris-HCl. |
publishDate |
2012 |
dc.date.available.fl_str_mv |
2012-10-03 2016-06-02T19:56:48Z |
dc.date.issued.fl_str_mv |
2012-03-30 |
dc.date.accessioned.fl_str_mv |
2016-06-02T19:56:48Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
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info:eu-repo/semantics/masterThesis |
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masterThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
VESCOVI, Vinicius. Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres. 2012. 93 f. Dissertação (Mestrado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2012. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/4099 |
identifier_str_mv |
VESCOVI, Vinicius. Extração, purificação e imobilização de lipases vegetais destinadas à síntese de biodiesel e ésteres. 2012. 93 f. Dissertação (Mestrado em Ciências Exatas e da Terra) - Universidade Federal de São Carlos, São Carlos, 2012. |
url |
https://repositorio.ufscar.br/handle/ufscar/4099 |
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openAccess |
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Universidade Federal de São Carlos |
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Programa de Pós-Graduação em Engenharia Química - PPGEQ |
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UFSCar |
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BR |
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Universidade Federal de São Carlos |
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