Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii
Autor(a) principal: | |
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Data de Publicação: | 2018 |
Tipo de documento: | Dissertação |
Idioma: | por |
Título da fonte: | Biblioteca Digital de Teses e Dissertações da UCB |
Texto Completo: | https://bdtd.ucb.br:8443/jspui/handle/tede/2406 |
Resumo: | Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study. |
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Biblioteca Digital de Teses e Dissertações da UCB |
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Parachin, N??dia Skorupahttp://lattes.cnpq.br/1214716137780644http://lattes.cnpq.br/6004547577932290Costa, Tha??s Duarte2018-06-06T14:01:33Z2018-04-03COSTA, Tha??s Duarte. Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii. 2018. 74 f. Disserta????o (Programa Stricto Sensu em Ci??ncias Gen??micas e Biotecnologia) - Universidade Cat??lica de Bras??lia, Bras??lia, 2018.https://bdtd.ucb.br:8443/jspui/handle/tede/2406Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study.Os problemas ambientais gerados por pl??sticos ?? base de tereftalato de polietileno (PET) se devem ao extenso tempo de decomposi????o desses materiais no meio ambiente e a sua fonte de origem que ?? dependente de petr??leo. Diante disso, biopl??sticos t??m sido uma alternativa promissora devido ao fato de serem biologicamente degrad??veis, al??m de terem como origem mat??rias-primas renov??veis, o que os tornam sustent??veis. Um dos pol??meros mais relevantes desse cen??rio ?? o poli (??cido l??tico) (PLA) formado a partir de mon??meros de ??cido l??tico. As principais caracter??sticas do PLA s??o baixa toxicidade aos humanos devido ?? alta biocompatibilidade, como por exemplo em mat??rias biom??dicos, e biodegradabilidade, o que reduz seu tempo em aterros devido ao processo mais r??pido de decomposi????o. Essas propriedades proporcionam uma ampla aplicabilidade deste pol??mero em diversas ??reas como embalagens, ??reas t??xteis e materiais biom??dicos. Comumente, o processo qu??mico de polimeriza????o do PLA pode ser realizado por meio de duas formas, (1) abertura do anel para posterior polimeriza????o ou (2) por condensa????o dos ??cidos l??ticos. Nos dois casos, ?? necess??ria a presen??a de catalisadores met??licos como zinco, alum??nio e magn??sio. Estes, al??m de serem t??xicos atrapalham na utiliza????o do pol??mero, por exemplo, na ??rea biom??dica, por gerar res??duos met??licos. Uma alternativa a esses catalisadores ?? a utiliza????o de biocatalisadores, como a polihidroxialcanoato sintase (phaC), j?? foi previamente utilizada para polimeriza????o de ??cido l??tico produzido em cepas recombinantes de Escherichia coli. Assim, dentro da plataforma de produ????o de ??cido l??tico, em cepas de Komagataella phaffii recombinantes, o objetivo deste trabalho ?? referente ?? produ????o da enzima phaC com muta????es pontuais nos s??tios S325N e Q481I, pois essas altera????es proporcionam uma maior especificidade do complexo enzima-substrato, para que atue como biocatalisador na polimeriza????o de ??cido l??tico em Komagataella phaffi. Neste estudo, foram realizadas tr??s estrat??gias de clonagem entre o inserto phaCPs e vetor pGAPZ??B. At?? o presente, n??o houve transformantes em nenhuma das estrat??gias. Entretanto, a Estrat??gia C ainda n??o foi executada completamente, o que resulta ainda na possibilidade de clonagem entre inserto phaCPs e vetor de express??o pGAPZ??B com a sequ??ncia correta. A expectativa deste estudo ?? a conclus??o da clonagem, verifica????o da sequ??ncia correta do DNA recombinante atrav??s do resultado do sequenciamento e inser????o do plasm??deo ao genoma da levedura Komagataella phaffii.Submitted by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:03Z No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5)Approved for entry into archive by Sara Ribeiro (sara.ribeiro@ucb.br) on 2018-06-06T14:01:33Z (GMT) No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5)Made available in DSpace on 2018-06-06T14:01:33Z (GMT). No. of bitstreams: 1 ThaisDuarteCostaDissertacao2018.pdf: 3076865 bytes, checksum: 13af7d694f07d7e2dcc9281907285b62 (MD5) Previous issue date: 2018-04-03application/pdfhttps://bdtd.ucb.br:8443/jspui/retrieve/5686/ThaisDuarteCostaDissertacao2018.pdf.jpgporUniversidade Cat??lica de Bras??liaPrograma Stricto Sensu em Ci??ncias Gen??micas e BiotecnologiaUCBBrasilEscola de Sa??de e MedicinaBiopl??sticoBiocatalisadorKomagataella phaffiiPoli (??cido l??tico) (PLA)BioplasticPolihidroxialcanoato sintase (phaC)Polyhydroxyalkanoate synthase (phaC)Poly lactic acid (PLA)BiocatalystCNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICAProdu????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffiiinfo:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisinfo:eu-repo/semantics/openAccessreponame:Biblioteca Digital de Teses e Dissertações da UCBinstname:Universidade Católica de Brasília (UCB)instacron:UCBLICENSElicense.txtlicense.txttext/plain; charset=utf-81905https://200.214.135.178:8443/jspui/bitstream/tede/2406/1/license.txt75558dcf859532757239878b42f1c2c7MD51ORIGINALThaisDuarteCostaDissertacao2018.pdfThaisDuarteCostaDissertacao2018.pdfapplication/pdf3076865https://200.214.135.178:8443/jspui/bitstream/tede/2406/2/ThaisDuarteCostaDissertacao2018.pdf13af7d694f07d7e2dcc9281907285b62MD52TEXTThaisDuarteCostaDissertacao2018.pdf.txtThaisDuarteCostaDissertacao2018.pdf.txttext/plain131745https://200.214.135.178:8443/jspui/bitstream/tede/2406/3/ThaisDuarteCostaDissertacao2018.pdf.txt3320d12f8c08e5f37818c73c7cf6eb2cMD53THUMBNAILThaisDuarteCostaDissertacao2018.pdf.jpgThaisDuarteCostaDissertacao2018.pdf.jpgimage/jpeg5803https://200.214.135.178:8443/jspui/bitstream/tede/2406/4/ThaisDuarteCostaDissertacao2018.pdf.jpg3a7652fa61b10181d8a9497f68781ed6MD54tede/24062018-06-07 11:26:07.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 Digital de Teses e Dissertaçõeshttps://bdtd.ucb.br:8443/jspui/ |
dc.title.por.fl_str_mv |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
title |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
spellingShingle |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii Costa, Tha??s Duarte Biopl??stico Biocatalisador Komagataella phaffii Poli (??cido l??tico) (PLA) BioplasticPolihidroxialcanoato sintase (phaC) Polyhydroxyalkanoate synthase (phaC) Poly lactic acid (PLA) Biocatalyst CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
title_short |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
title_full |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
title_fullStr |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
title_full_unstemmed |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
title_sort |
Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii |
author |
Costa, Tha??s Duarte |
author_facet |
Costa, Tha??s Duarte |
author_role |
author |
dc.contributor.advisor1.fl_str_mv |
Parachin, N??dia Skorupa |
dc.contributor.advisor1Lattes.fl_str_mv |
http://lattes.cnpq.br/1214716137780644 |
dc.contributor.authorLattes.fl_str_mv |
http://lattes.cnpq.br/6004547577932290 |
dc.contributor.author.fl_str_mv |
Costa, Tha??s Duarte |
contributor_str_mv |
Parachin, N??dia Skorupa |
dc.subject.por.fl_str_mv |
Biopl??stico Biocatalisador Komagataella phaffii Poli (??cido l??tico) (PLA) BioplasticPolihidroxialcanoato sintase (phaC) Polyhydroxyalkanoate synthase (phaC) Poly lactic acid (PLA) Biocatalyst |
topic |
Biopl??stico Biocatalisador Komagataella phaffii Poli (??cido l??tico) (PLA) BioplasticPolihidroxialcanoato sintase (phaC) Polyhydroxyalkanoate synthase (phaC) Poly lactic acid (PLA) Biocatalyst CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.subject.cnpq.fl_str_mv |
CNPQ::CIENCIAS BIOLOGICAS::BIOQUIMICA |
dc.description.abstract.eng.fl_txt_mv |
Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study. |
dc.description.abstract.por.fl_txt_mv |
Os problemas ambientais gerados por pl??sticos ?? base de tereftalato de polietileno (PET) se devem ao extenso tempo de decomposi????o desses materiais no meio ambiente e a sua fonte de origem que ?? dependente de petr??leo. Diante disso, biopl??sticos t??m sido uma alternativa promissora devido ao fato de serem biologicamente degrad??veis, al??m de terem como origem mat??rias-primas renov??veis, o que os tornam sustent??veis. Um dos pol??meros mais relevantes desse cen??rio ?? o poli (??cido l??tico) (PLA) formado a partir de mon??meros de ??cido l??tico. As principais caracter??sticas do PLA s??o baixa toxicidade aos humanos devido ?? alta biocompatibilidade, como por exemplo em mat??rias biom??dicos, e biodegradabilidade, o que reduz seu tempo em aterros devido ao processo mais r??pido de decomposi????o. Essas propriedades proporcionam uma ampla aplicabilidade deste pol??mero em diversas ??reas como embalagens, ??reas t??xteis e materiais biom??dicos. Comumente, o processo qu??mico de polimeriza????o do PLA pode ser realizado por meio de duas formas, (1) abertura do anel para posterior polimeriza????o ou (2) por condensa????o dos ??cidos l??ticos. Nos dois casos, ?? necess??ria a presen??a de catalisadores met??licos como zinco, alum??nio e magn??sio. Estes, al??m de serem t??xicos atrapalham na utiliza????o do pol??mero, por exemplo, na ??rea biom??dica, por gerar res??duos met??licos. Uma alternativa a esses catalisadores ?? a utiliza????o de biocatalisadores, como a polihidroxialcanoato sintase (phaC), j?? foi previamente utilizada para polimeriza????o de ??cido l??tico produzido em cepas recombinantes de Escherichia coli. Assim, dentro da plataforma de produ????o de ??cido l??tico, em cepas de Komagataella phaffii recombinantes, o objetivo deste trabalho ?? referente ?? produ????o da enzima phaC com muta????es pontuais nos s??tios S325N e Q481I, pois essas altera????es proporcionam uma maior especificidade do complexo enzima-substrato, para que atue como biocatalisador na polimeriza????o de ??cido l??tico em Komagataella phaffi. Neste estudo, foram realizadas tr??s estrat??gias de clonagem entre o inserto phaCPs e vetor pGAPZ??B. At?? o presente, n??o houve transformantes em nenhuma das estrat??gias. Entretanto, a Estrat??gia C ainda n??o foi executada completamente, o que resulta ainda na possibilidade de clonagem entre inserto phaCPs e vetor de express??o pGAPZ??B com a sequ??ncia correta. A expectativa deste estudo ?? a conclus??o da clonagem, verifica????o da sequ??ncia correta do DNA recombinante atrav??s do resultado do sequenciamento e inser????o do plasm??deo ao genoma da levedura Komagataella phaffii. |
description |
Polyethylene terephthalate (PET) based plastics are serious environmental problem due to long decomposition periods and petroleum-dependent origin. Therefore, bioplastics are a promising alternative as their synthesized by the polimerization of renewable raw materials, yeilding biodegradable and environmental-friendly products. One of the most relevant polymers in this scenario is the poly lactic acid (PLA) formed from lactic acid monomers. The main characteristics of PLA are low toxicity to humans due to high biocompatibility, for example in biomedical materials, and biodegradability, which reduces their time in landfills due to the faster decomposition process. These properties provide wide applicability of this polymer in various areas such as packaging, textiles and biomedical materials. Commonly, the chemical polymerization process of PLA can be carried out in two ways, (1) ring opening for further polymerization or (2) condensation of the lactic acids. In both cases, the presence of metal catalysts such as zinc, aluminum and magnesium is required. These, in addition to being toxic, hinder the use of the polymer, for instance, in the biomedical area, for generating metallic waste. An alternative to such catalysts is the use of biocatalysts. Polyhydroxyalkanoate synthase (phaC) has been previously used for the polymerization of lactic acid produced in recombinant strains of Escherichia coli. Thus, within the lactic acid production platform in recombinant Komagataella phaffi strains, the objective of this work is to produce the phaC enzyme with point mutations at the S325N and Q481I sites. These residue changes provide a greater specificity of the enzyme-substrate complex to act as a biocatalyst in the polymerization of lactic acid in Komagataella phaffi. In this study, three cloning strategies were performed between the phaCPs insert and pGAPZ??B vector. To date, there have been no transformants in any of the strategies. However, Strategy C has not yet been fully implemented, which also results in the possibility of cloning between phaCPs insert and pGAPZ??B expression vector with the correct sequence. It is expected that successful cloning, recombinant DNA sequencing and plasmid insertion into Komagataella phaffii genome can be performed to conclude this study. |
publishDate |
2018 |
dc.date.accessioned.fl_str_mv |
2018-06-06T14:01:33Z |
dc.date.issued.fl_str_mv |
2018-04-03 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/masterThesis |
status_str |
publishedVersion |
format |
masterThesis |
dc.identifier.citation.fl_str_mv |
COSTA, Tha??s Duarte. Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii. 2018. 74 f. Disserta????o (Programa Stricto Sensu em Ci??ncias Gen??micas e Biotecnologia) - Universidade Cat??lica de Bras??lia, Bras??lia, 2018. |
dc.identifier.uri.fl_str_mv |
https://bdtd.ucb.br:8443/jspui/handle/tede/2406 |
identifier_str_mv |
COSTA, Tha??s Duarte. Produ????o heter??loga de polihidroxialcanoato sintase (PhaC), biocatalisador da s??ntese de Poli (??cido l??tico) (PLA) em Komagataella phaffii. 2018. 74 f. Disserta????o (Programa Stricto Sensu em Ci??ncias Gen??micas e Biotecnologia) - Universidade Cat??lica de Bras??lia, Bras??lia, 2018. |
url |
https://bdtd.ucb.br:8443/jspui/handle/tede/2406 |
dc.language.iso.fl_str_mv |
por |
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por |
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info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
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Universidade Cat??lica de Bras??lia |
dc.publisher.program.fl_str_mv |
Programa Stricto Sensu em Ci??ncias Gen??micas e Biotecnologia |
dc.publisher.initials.fl_str_mv |
UCB |
dc.publisher.country.fl_str_mv |
Brasil |
dc.publisher.department.fl_str_mv |
Escola de Sa??de e Medicina |
publisher.none.fl_str_mv |
Universidade Cat??lica de Bras??lia |
dc.source.none.fl_str_mv |
reponame:Biblioteca Digital de Teses e Dissertações da UCB instname:Universidade Católica de Brasília (UCB) instacron:UCB |
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Universidade Católica de Brasília (UCB) |
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UCB |
institution |
UCB |
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Biblioteca Digital de Teses e Dissertações da UCB |
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Biblioteca Digital de Teses e Dissertações da UCB |
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