Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal
Autor(a) principal: | |
---|---|
Data de Publicação: | 2015 |
Tipo de documento: | Tese |
Idioma: | por |
Título da fonte: | Repositório Institucional da UFSCAR |
Texto Completo: | https://repositorio.ufscar.br/handle/ufscar/279 |
Resumo: | Currently the great challenge for the production of second generation ethanol is to reduce thecost of the enzymes. It is possible to reduce part of this cost by carrying out an optimization ofthe process fermentation using sources of cellulase-induction that allow further growth of fungalbiomass, and increased secretion of proteins. The use of a mutant strain with overexpression ofhemicelulolytics activators could increase expression of the enzymes of interest and therebycontribute to cost reduction. This work aimed to study the production of enzymes involved inthe degradation of biomass by the newly isolated strain of Trichoderma harzianum P49P11focusing on the improvement of the submerged fermentation processes and on the use molecularbiology tools to improve the fungal strain. Regarding the fermentation process, the effects ofdifferent inducing sources were evaluated in flasks and bioreactor using statistical experimentaldesign tools and strategies to enhance biomass in the pre-culture step. For fungal strainimprovement, it was used molecular biology tools for the overexpression of two activators ofcellulases (xyr1 and lae1). A proteomic analysis of the T. harzianum enzymatic extract obtainedusing sugarcane bagasse pretreated by steam explosion followed by delignification (BED) wasperformed. The results showed that the best source for inducing cellulase was BED + sucrose(3: 1), reaching values of 1.21 FPU/mL 80.0U/mL of xylanase and 17.30 U/mL of β-glucosidase. The proteomic analyis identificated 24 different glycoside hydrolases and fourCBM proteins, within 12 different CAZy families. From this study, the enzymatic cocktailproduced "on site" could be supplemented using two accessory enzymes, pectinase and α-Larabinofuranosidase,leading to an increase of 100% of the hydrolysis yield. Regarding thestudy of carbon sources in the pre-culture step, it was possible to increase cellulases productionin 2 times using glycerol as the initial carbon source, followed by inducing carbon source(BED). The xyr1 and lae1 overexpression influencied positively the FPase, CMCase, xylanaseand β-glucosidase production, representing a new approach to increase production of theseenzymes. |
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Delabona, Priscila da SilvaFarinas, Cristiane Sanchezhttp://lattes.cnpq.br/9933650905615452http://lattes.cnpq.br/9016348395509598f0e1fd85-6ba4-4265-836a-f14bde32bcc02016-06-02T19:02:45Z2015-04-222016-06-02T19:02:45Z2015-03-20DELABONA, Priscila da Silva. Production of cellulases by Trichoderma harzianum for biomass process to bioethanol. 2015. 150 f. Tese (Doutorado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2015.https://repositorio.ufscar.br/handle/ufscar/279Currently the great challenge for the production of second generation ethanol is to reduce thecost of the enzymes. It is possible to reduce part of this cost by carrying out an optimization ofthe process fermentation using sources of cellulase-induction that allow further growth of fungalbiomass, and increased secretion of proteins. The use of a mutant strain with overexpression ofhemicelulolytics activators could increase expression of the enzymes of interest and therebycontribute to cost reduction. This work aimed to study the production of enzymes involved inthe degradation of biomass by the newly isolated strain of Trichoderma harzianum P49P11focusing on the improvement of the submerged fermentation processes and on the use molecularbiology tools to improve the fungal strain. Regarding the fermentation process, the effects ofdifferent inducing sources were evaluated in flasks and bioreactor using statistical experimentaldesign tools and strategies to enhance biomass in the pre-culture step. For fungal strainimprovement, it was used molecular biology tools for the overexpression of two activators ofcellulases (xyr1 and lae1). A proteomic analysis of the T. harzianum enzymatic extract obtainedusing sugarcane bagasse pretreated by steam explosion followed by delignification (BED) wasperformed. The results showed that the best source for inducing cellulase was BED + sucrose(3: 1), reaching values of 1.21 FPU/mL 80.0U/mL of xylanase and 17.30 U/mL of β-glucosidase. The proteomic analyis identificated 24 different glycoside hydrolases and fourCBM proteins, within 12 different CAZy families. From this study, the enzymatic cocktailproduced "on site" could be supplemented using two accessory enzymes, pectinase and α-Larabinofuranosidase,leading to an increase of 100% of the hydrolysis yield. Regarding thestudy of carbon sources in the pre-culture step, it was possible to increase cellulases productionin 2 times using glycerol as the initial carbon source, followed by inducing carbon source(BED). The xyr1 and lae1 overexpression influencied positively the FPase, CMCase, xylanaseand β-glucosidase production, representing a new approach to increase production of theseenzymes.Atualmente o grande desafio para a produção de etanol de segunda geração consiste emdiminuir o custo das enzimas degradantes da fibra lignocelulósica. Uma alternativa quepode levar a redução de parte desse custo é a otimização do processo fermentativoutilizando fontes indutoras das enzimas (hemi)celulolíticas que permitam um maiorcrescimento da biomassa fúngica e maior secreção de proteínas. A utilização de umalinhagem mutante com a superexpressão dos ativadores dessas enzimas também podecontribuir nesse sentido. Portanto, o objetivo deste trabalho foi estudar a produção deenzimas envolvidas na degradação da biomassa pelo fungo recém-isolado Trichodermaharzianum P49P11 com foco no melhoramento dos processos fermentativos submersose no uso de ferramentas de biologia molecular para o melhoramento da linhagemfúngica. Em relação ao processo fermentativo foi avaliado o efeito de diferentes fontesindutoras em frascos agitados e em biorreator utilizando ferramentas estatísticas deplanejamento de experimentos e estratégias de aumento de biomassa na fase de préinóculo.Em relação ao melhoramento da linhagem fungica foram utilizadas ferramentasde biologia molecular para a superexpressão de dois ativadores de celulases (xyr1 elae1). Para um melhor entendimento das proteínas produzidas em cultivo submerso foirealizado o proteôma do extrato enzimático secretado pelo fungo selvagem em bagaçode cana pré-tratado. Os resultados mostraram que a melhor fonte de carbono indutora decelulases foi o bagaço de cana explodido e deslignificado (BED) + sacarose naproporção 3:1, alcançando valores de 1,21 FPU/mL, 80.0U/mL de xilanse e 17,30 U/mL de β-glicosidase. O proteôma do extrato enzimático de T. harzianum resultou naidentificação de 24 hidrolases glicosídicas diferentes, 4 proteínas CBM dentro de 12diferentes famílias do CAZy. A partir deste estudo pode-se suplementar o coquetelenzimático on site com duas enzimas acessórias, pectinase e α-L-arabinofuranosidaseque aumentaram o rendimento de hidrólise em mais de 100%. Em relação ao estudo dasfontes de carbono na fase de pré-inóculo foi possível aumentar a produção de celulasesem 130% utilizando o glicerol como fonte de carbono inicial, seguida de fonte de18carbono indutora (BED). A aplicação de técnicas de biologia molecular para a mutaçãodo T. harzianum visando a superexpressão de xyr1 e lae teve influência positiva naprodução das enzimas FPase, CMCase, xilanase e β-glicosidase, representando umanova abordagem para aumentar a produção dessas enzimas.Financiadora de Estudos e Projetosapplication/pdfporUniversidade Federal de São CarlosPrograma de Pós-Graduação em Biotecnologia - PPGBiotecUFSCarBRBiotecnologiaTrichoderma harzianumBiomassa vegetalCelulaseTrichoderma harzianumCellulasesInductors and cellulases activatorsOUTROSProdução de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetalProduction of cellulases by Trichoderma harzianum for biomass process to bioethanolinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/doctoralThesis-1-1dd9ceb6c-d509-421a-a31e-bcb55bb21e02info:eu-repo/semantics/openAccessreponame:Repositório Institucional da UFSCARinstname:Universidade Federal de São Carlos (UFSCAR)instacron:UFSCARORIGINAL6663.pdfapplication/pdf3464569https://repositorio.ufscar.br/bitstream/ufscar/279/1/6663.pdf56765698cbf2bedabdd5b1e555a404aeMD51TEXT6663.pdf.txt6663.pdf.txtExtracted texttext/plain0https://repositorio.ufscar.br/bitstream/ufscar/279/2/6663.pdf.txtd41d8cd98f00b204e9800998ecf8427eMD52THUMBNAIL6663.pdf.jpg6663.pdf.jpgIM Thumbnailimage/jpeg7814https://repositorio.ufscar.br/bitstream/ufscar/279/3/6663.pdf.jpg4fe5e95894e01ee582046d5ab6fca516MD53ufscar/2792023-09-18 18:31:27.122oai:repositorio.ufscar.br:ufscar/279Repositório InstitucionalPUBhttps://repositorio.ufscar.br/oai/requestopendoar:43222023-09-18T18:31:27Repositório Institucional da UFSCAR - Universidade Federal de São Carlos (UFSCAR)false |
dc.title.por.fl_str_mv |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
dc.title.alternative.eng.fl_str_mv |
Production of cellulases by Trichoderma harzianum for biomass process to bioethanol |
title |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
spellingShingle |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal Delabona, Priscila da Silva Biotecnologia Trichoderma harzianum Biomassa vegetal Celulase Trichoderma harzianum Cellulases Inductors and cellulases activators OUTROS |
title_short |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
title_full |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
title_fullStr |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
title_full_unstemmed |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
title_sort |
Produção de glicosil hidrolases por Trichoderma harzianum para o processo de sacarificação da biomassa vegetal |
author |
Delabona, Priscila da Silva |
author_facet |
Delabona, Priscila da Silva |
author_role |
author |
dc.contributor.authorlattes.por.fl_str_mv |
http://lattes.cnpq.br/9016348395509598 |
dc.contributor.author.fl_str_mv |
Delabona, Priscila da Silva |
dc.contributor.advisor1.fl_str_mv |
Farinas, Cristiane Sanchez |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/9933650905615452 |
dc.contributor.authorID.fl_str_mv |
f0e1fd85-6ba4-4265-836a-f14bde32bcc0 |
contributor_str_mv |
Farinas, Cristiane Sanchez |
dc.subject.por.fl_str_mv |
Biotecnologia Trichoderma harzianum Biomassa vegetal Celulase |
topic |
Biotecnologia Trichoderma harzianum Biomassa vegetal Celulase Trichoderma harzianum Cellulases Inductors and cellulases activators OUTROS |
dc.subject.eng.fl_str_mv |
Trichoderma harzianum Cellulases Inductors and cellulases activators |
dc.subject.cnpq.fl_str_mv |
OUTROS |
description |
Currently the great challenge for the production of second generation ethanol is to reduce thecost of the enzymes. It is possible to reduce part of this cost by carrying out an optimization ofthe process fermentation using sources of cellulase-induction that allow further growth of fungalbiomass, and increased secretion of proteins. The use of a mutant strain with overexpression ofhemicelulolytics activators could increase expression of the enzymes of interest and therebycontribute to cost reduction. This work aimed to study the production of enzymes involved inthe degradation of biomass by the newly isolated strain of Trichoderma harzianum P49P11focusing on the improvement of the submerged fermentation processes and on the use molecularbiology tools to improve the fungal strain. Regarding the fermentation process, the effects ofdifferent inducing sources were evaluated in flasks and bioreactor using statistical experimentaldesign tools and strategies to enhance biomass in the pre-culture step. For fungal strainimprovement, it was used molecular biology tools for the overexpression of two activators ofcellulases (xyr1 and lae1). A proteomic analysis of the T. harzianum enzymatic extract obtainedusing sugarcane bagasse pretreated by steam explosion followed by delignification (BED) wasperformed. The results showed that the best source for inducing cellulase was BED + sucrose(3: 1), reaching values of 1.21 FPU/mL 80.0U/mL of xylanase and 17.30 U/mL of β-glucosidase. The proteomic analyis identificated 24 different glycoside hydrolases and fourCBM proteins, within 12 different CAZy families. From this study, the enzymatic cocktailproduced "on site" could be supplemented using two accessory enzymes, pectinase and α-Larabinofuranosidase,leading to an increase of 100% of the hydrolysis yield. Regarding thestudy of carbon sources in the pre-culture step, it was possible to increase cellulases productionin 2 times using glycerol as the initial carbon source, followed by inducing carbon source(BED). The xyr1 and lae1 overexpression influencied positively the FPase, CMCase, xylanaseand β-glucosidase production, representing a new approach to increase production of theseenzymes. |
publishDate |
2015 |
dc.date.available.fl_str_mv |
2015-04-22 2016-06-02T19:02:45Z |
dc.date.issued.fl_str_mv |
2015-03-20 |
dc.date.accessioned.fl_str_mv |
2016-06-02T19:02:45Z |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/doctoralThesis |
format |
doctoralThesis |
status_str |
publishedVersion |
dc.identifier.citation.fl_str_mv |
DELABONA, Priscila da Silva. Production of cellulases by Trichoderma harzianum for biomass process to bioethanol. 2015. 150 f. Tese (Doutorado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2015. |
dc.identifier.uri.fl_str_mv |
https://repositorio.ufscar.br/handle/ufscar/279 |
identifier_str_mv |
DELABONA, Priscila da Silva. Production of cellulases by Trichoderma harzianum for biomass process to bioethanol. 2015. 150 f. Tese (Doutorado em Multidisciplinar) - Universidade Federal de São Carlos, São Carlos, 2015. |
url |
https://repositorio.ufscar.br/handle/ufscar/279 |
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por |
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dd9ceb6c-d509-421a-a31e-bcb55bb21e02 |
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info:eu-repo/semantics/openAccess |
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openAccess |
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application/pdf |
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Universidade Federal de São Carlos |
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Programa de Pós-Graduação em Biotecnologia - PPGBiotec |
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UFSCar |
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BR |
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Universidade Federal de São Carlos |
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