Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid

Detalhes bibliográficos
Autor(a) principal: Anayla dos Santos Sousa
Data de Publicação: 2007
Tipo de documento: Dissertação
Idioma: por
Título da fonte: Biblioteca Digital de Teses e Dissertações da UFC
Texto Completo: http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2410
Resumo: Hyaluronic acid (HA) is a linear, unbranched polymer, composed by simple disaccharide units, whose molar mass may range in a wide interval of distribution (104-107 Da). It is used in pharmaceutical and cosmetic applications depending on its average molecular mass. The fractionation of macromolecules of the order of magnitude of the HA may be performed by gel permeation or size-exclusion chromatography (GPC). The objective of this work is to study the characterization, purification and fractionation of microbial HA by using GPC. HA was supplied by the Department of Biotechnological Processes (DPB/FEQ/UNICAMP), having been produced by microorganisms â Streptococcus zooepidemicus â in synthetic culture media. After being centrifuged from the fermentation broth, HA was precipitated with ethanol and then dissolved in saline solution, this pre-purification procedure being performed up to four times. The presence of proteic contaminants was identified by electrophoresis in polyacrylamide gel under denaturant conditions (SDS-PAGE) and quantified by the Bradford method. For the HA sample which had been precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, protein concentration was found to be nearly 100 μg/mL. The sodium salt of commercial (Sigma Aldrich - EUA) microbial HA was used as standard in order to measure a calibration curve to assess HA concentration in the samples. For the sample precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, AH concentration was found to be approximately 780 μg/mL. Molar mass distribution of HA samples was assessed by GPC, both in analytical and semi-preparative scale, using refractive index (RI) and UV/Vis (280 nm) detectors, 0.1 M NaNO3 as mobile phase, flow rate 0.8 mL/min and tracers dextran and pullulan (polymers of similar hydrodynamic volume as compared to HA) as molar mass standards. The samples showed molar mass distributions in the range of 103 to 107 Da, after being precipitated and dissolved four times. For these samples, the average molar mass was found to be in the order of magnitude of 105 Da. In the semi-preparative column Superose, it was possible to separate HA fractions, with molar mass above 105 Da, and free of proteic contaminants, in an elution interval of 10 minutes
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spelling info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/masterThesisGel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acidCromatografia de permeaÃÃo em gel em escala semi-preservativa aplicada à caracterizaÃÃo, purificaÃÃo e fracionamento do Ãcido hialurÃnico produzido por cultivo de microorganismos2007-03-20Diana Cristina Silva de Azevedo31021409391Josà Soares de Andrade JÃnior43946145400Cesar Costapinto Santana20541430734http://lattes.cnpq.br/8458559657676452 Maria Helena Andrade Santana0162451385982287961372Anayla dos Santos SousaUniversidade Federal do CearÃPrograma de PÃs-GraduaÃÃo em Engenharia QuÃmicaUFCBRÃcido hialurÃnico Cromatografia de PermeaÃÃo em Gel PurificaÃÃo Massa MolarHyaluronic Acid Gel Permeation Chromatography Purification Molar MassENGENHARIA QUIMICAHyaluronic acid (HA) is a linear, unbranched polymer, composed by simple disaccharide units, whose molar mass may range in a wide interval of distribution (104-107 Da). It is used in pharmaceutical and cosmetic applications depending on its average molecular mass. The fractionation of macromolecules of the order of magnitude of the HA may be performed by gel permeation or size-exclusion chromatography (GPC). The objective of this work is to study the characterization, purification and fractionation of microbial HA by using GPC. HA was supplied by the Department of Biotechnological Processes (DPB/FEQ/UNICAMP), having been produced by microorganisms â Streptococcus zooepidemicus â in synthetic culture media. After being centrifuged from the fermentation broth, HA was precipitated with ethanol and then dissolved in saline solution, this pre-purification procedure being performed up to four times. The presence of proteic contaminants was identified by electrophoresis in polyacrylamide gel under denaturant conditions (SDS-PAGE) and quantified by the Bradford method. For the HA sample which had been precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, protein concentration was found to be nearly 100 μg/mL. The sodium salt of commercial (Sigma Aldrich - EUA) microbial HA was used as standard in order to measure a calibration curve to assess HA concentration in the samples. For the sample precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, AH concentration was found to be approximately 780 μg/mL. Molar mass distribution of HA samples was assessed by GPC, both in analytical and semi-preparative scale, using refractive index (RI) and UV/Vis (280 nm) detectors, 0.1 M NaNO3 as mobile phase, flow rate 0.8 mL/min and tracers dextran and pullulan (polymers of similar hydrodynamic volume as compared to HA) as molar mass standards. The samples showed molar mass distributions in the range of 103 to 107 Da, after being precipitated and dissolved four times. For these samples, the average molar mass was found to be in the order of magnitude of 105 Da. In the semi-preparative column Superose, it was possible to separate HA fractions, with molar mass above 105 Da, and free of proteic contaminants, in an elution interval of 10 minutesO Ãcido HialurÃnico (AH) à um polÃmero linear, nÃo ramificado, composto por unidades dissacarÃdicas simples, cuja massa molar pode se distribuir num largo intervalo (104-107 Da). à usado em aplicaÃÃes farmacÃuticas e cosmÃticas dependendo de sua massa molar mÃdia. A separaÃÃo de macromolÃculas da ordem de grandeza do AH à possÃvel atravÃs da Cromatografia de permeaÃÃo em gel (GPC). Este trabalho tem como objetivo estudar a caracterizaÃÃo, purificaÃÃo e fracionamento do AH produzido por microorganismos, utilizando GPC. O AH, cedido pelo Departamento de Processos BiotecnolÃgicos (DPB/FEQ/UNICAMP), foi obtido por cultivo de microorganismos - Streptococcus zooepidemicus - em meio de cultura sintÃtico. ApÃs centrifugaÃÃo do caldo de fermentaÃÃo, o AH foi precipitado com etanol e ressuspenso em soluÃÃo salina, sendo esta etapa de prÃ-purificaÃÃo executada atà quatro vezes. A presenÃa de contaminantes protÃicos foi identificada por eletroforese em gel de poliacrilamida sob condiÃÃes desnaturantes (SDS-PAGE) e quantificada pelo mÃtodo de Bradford, obtendo-se 97,20 μg/mL, para a amostra de AH apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M. Para as determinaÃÃes de concentraÃÃo de AH nas amostras estudadas, utilizou-se o sal sÃdico de AH microbial comercial (Sigma Aldrich - EUA) como padrÃo. Quantificando-se a amostra de AH apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M, obteve-se 783,70 μg/mL. A distribuiÃÃo da massa molar do AH foi avaliada por GPC, em escala analÃtica, utilizando coluna Shodex OHPak-SB806M-HQ e, em escala semi-preparativa, a coluna Superose 6, detectores de Ãndice de refraÃÃo e UV/Vis (280 nm), NaNO3 0,1 M como fase mÃvel, vazÃo de 0,8 mL/min e padrÃes de Dextrana e Pullulan (polÃmeros de volume hidrodinÃmico semelhante ao AH) como marcadores de massa molar (5,2x103 - 7,9x106 Da e 5,8x103 â 8,53x105 Da, respectivamente). A amostra apresentou uma distribuiÃÃo polimÃrica na faixa de 103 a 107 Da, apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M, sendo sua massa molar mÃdia da ordem de 105 Da. Foi possÃvel separar fraÃÃes de AH, acima de 105 Da, livre de contaminantes protÃicos, num intervalo de aproximadamente 10 minutos, na coluna Superose 6CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superiorhttp://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2410application/pdfinfo:eu-repo/semantics/openAccessporreponame:Biblioteca Digital de Teses e Dissertações da UFCinstname:Universidade Federal do Cearáinstacron:UFC2019-01-21T11:15:31Zmail@mail.com -
dc.title.en.fl_str_mv Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
dc.title.alternative.pt.fl_str_mv Cromatografia de permeaÃÃo em gel em escala semi-preservativa aplicada à caracterizaÃÃo, purificaÃÃo e fracionamento do Ãcido hialurÃnico produzido por cultivo de microorganismos
title Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
spellingShingle Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
Anayla dos Santos Sousa
Ãcido hialurÃnico
Cromatografia de PermeaÃÃo em Gel
PurificaÃÃo
Massa Molar
Hyaluronic Acid
Gel Permeation Chromatography
Purification
Molar Mass
ENGENHARIA QUIMICA
title_short Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
title_full Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
title_fullStr Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
title_full_unstemmed Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
title_sort Gel permeation chromatography in semi-preparative scale apply to the characterization, purification and fractionation of microbial hyaluronic acid
author Anayla dos Santos Sousa
author_facet Anayla dos Santos Sousa
author_role author
dc.contributor.advisor1.fl_str_mv Diana Cristina Silva de Azevedo
dc.contributor.advisor1ID.fl_str_mv 31021409391
dc.contributor.referee1.fl_str_mv Josà Soares de Andrade JÃnior
dc.contributor.referee1ID.fl_str_mv 43946145400
dc.contributor.referee2.fl_str_mv Cesar Costapinto Santana
dc.contributor.referee2ID.fl_str_mv 20541430734
dc.contributor.referee2Lattes.fl_str_mv http://lattes.cnpq.br/8458559657676452
dc.contributor.referee3.fl_str_mv Maria Helena Andrade Santana
dc.contributor.referee3ID.fl_str_mv 01624513859
dc.contributor.authorID.fl_str_mv 82287961372
dc.contributor.author.fl_str_mv Anayla dos Santos Sousa
contributor_str_mv Diana Cristina Silva de Azevedo
Josà Soares de Andrade JÃnior
Cesar Costapinto Santana
Maria Helena Andrade Santana
dc.subject.por.fl_str_mv Ãcido hialurÃnico
Cromatografia de PermeaÃÃo em Gel
PurificaÃÃo
Massa Molar
topic Ãcido hialurÃnico
Cromatografia de PermeaÃÃo em Gel
PurificaÃÃo
Massa Molar
Hyaluronic Acid
Gel Permeation Chromatography
Purification
Molar Mass
ENGENHARIA QUIMICA
dc.subject.eng.fl_str_mv Hyaluronic Acid
Gel Permeation Chromatography
Purification
Molar Mass
dc.subject.cnpq.fl_str_mv ENGENHARIA QUIMICA
dc.description.sponsorship.fl_txt_mv CoordenaÃÃo de AperfeiÃoamento de Pessoal de NÃvel Superior
dc.description.abstract.por.fl_txt_mv Hyaluronic acid (HA) is a linear, unbranched polymer, composed by simple disaccharide units, whose molar mass may range in a wide interval of distribution (104-107 Da). It is used in pharmaceutical and cosmetic applications depending on its average molecular mass. The fractionation of macromolecules of the order of magnitude of the HA may be performed by gel permeation or size-exclusion chromatography (GPC). The objective of this work is to study the characterization, purification and fractionation of microbial HA by using GPC. HA was supplied by the Department of Biotechnological Processes (DPB/FEQ/UNICAMP), having been produced by microorganisms â Streptococcus zooepidemicus â in synthetic culture media. After being centrifuged from the fermentation broth, HA was precipitated with ethanol and then dissolved in saline solution, this pre-purification procedure being performed up to four times. The presence of proteic contaminants was identified by electrophoresis in polyacrylamide gel under denaturant conditions (SDS-PAGE) and quantified by the Bradford method. For the HA sample which had been precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, protein concentration was found to be nearly 100 μg/mL. The sodium salt of commercial (Sigma Aldrich - EUA) microbial HA was used as standard in order to measure a calibration curve to assess HA concentration in the samples. For the sample precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, AH concentration was found to be approximately 780 μg/mL. Molar mass distribution of HA samples was assessed by GPC, both in analytical and semi-preparative scale, using refractive index (RI) and UV/Vis (280 nm) detectors, 0.1 M NaNO3 as mobile phase, flow rate 0.8 mL/min and tracers dextran and pullulan (polymers of similar hydrodynamic volume as compared to HA) as molar mass standards. The samples showed molar mass distributions in the range of 103 to 107 Da, after being precipitated and dissolved four times. For these samples, the average molar mass was found to be in the order of magnitude of 105 Da. In the semi-preparative column Superose, it was possible to separate HA fractions, with molar mass above 105 Da, and free of proteic contaminants, in an elution interval of 10 minutes
O Ãcido HialurÃnico (AH) à um polÃmero linear, nÃo ramificado, composto por unidades dissacarÃdicas simples, cuja massa molar pode se distribuir num largo intervalo (104-107 Da). à usado em aplicaÃÃes farmacÃuticas e cosmÃticas dependendo de sua massa molar mÃdia. A separaÃÃo de macromolÃculas da ordem de grandeza do AH à possÃvel atravÃs da Cromatografia de permeaÃÃo em gel (GPC). Este trabalho tem como objetivo estudar a caracterizaÃÃo, purificaÃÃo e fracionamento do AH produzido por microorganismos, utilizando GPC. O AH, cedido pelo Departamento de Processos BiotecnolÃgicos (DPB/FEQ/UNICAMP), foi obtido por cultivo de microorganismos - Streptococcus zooepidemicus - em meio de cultura sintÃtico. ApÃs centrifugaÃÃo do caldo de fermentaÃÃo, o AH foi precipitado com etanol e ressuspenso em soluÃÃo salina, sendo esta etapa de prÃ-purificaÃÃo executada atà quatro vezes. A presenÃa de contaminantes protÃicos foi identificada por eletroforese em gel de poliacrilamida sob condiÃÃes desnaturantes (SDS-PAGE) e quantificada pelo mÃtodo de Bradford, obtendo-se 97,20 μg/mL, para a amostra de AH apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M. Para as determinaÃÃes de concentraÃÃo de AH nas amostras estudadas, utilizou-se o sal sÃdico de AH microbial comercial (Sigma Aldrich - EUA) como padrÃo. Quantificando-se a amostra de AH apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M, obteve-se 783,70 μg/mL. A distribuiÃÃo da massa molar do AH foi avaliada por GPC, em escala analÃtica, utilizando coluna Shodex OHPak-SB806M-HQ e, em escala semi-preparativa, a coluna Superose 6, detectores de Ãndice de refraÃÃo e UV/Vis (280 nm), NaNO3 0,1 M como fase mÃvel, vazÃo de 0,8 mL/min e padrÃes de Dextrana e Pullulan (polÃmeros de volume hidrodinÃmico semelhante ao AH) como marcadores de massa molar (5,2x103 - 7,9x106 Da e 5,8x103 â 8,53x105 Da, respectivamente). A amostra apresentou uma distribuiÃÃo polimÃrica na faixa de 103 a 107 Da, apÃs quatro precipitaÃÃes em etanol e ressuspensÃes em NaNO3 0,1 M, sendo sua massa molar mÃdia da ordem de 105 Da. Foi possÃvel separar fraÃÃes de AH, acima de 105 Da, livre de contaminantes protÃicos, num intervalo de aproximadamente 10 minutos, na coluna Superose 6
description Hyaluronic acid (HA) is a linear, unbranched polymer, composed by simple disaccharide units, whose molar mass may range in a wide interval of distribution (104-107 Da). It is used in pharmaceutical and cosmetic applications depending on its average molecular mass. The fractionation of macromolecules of the order of magnitude of the HA may be performed by gel permeation or size-exclusion chromatography (GPC). The objective of this work is to study the characterization, purification and fractionation of microbial HA by using GPC. HA was supplied by the Department of Biotechnological Processes (DPB/FEQ/UNICAMP), having been produced by microorganisms â Streptococcus zooepidemicus â in synthetic culture media. After being centrifuged from the fermentation broth, HA was precipitated with ethanol and then dissolved in saline solution, this pre-purification procedure being performed up to four times. The presence of proteic contaminants was identified by electrophoresis in polyacrylamide gel under denaturant conditions (SDS-PAGE) and quantified by the Bradford method. For the HA sample which had been precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, protein concentration was found to be nearly 100 μg/mL. The sodium salt of commercial (Sigma Aldrich - EUA) microbial HA was used as standard in order to measure a calibration curve to assess HA concentration in the samples. For the sample precipitated in ethanol and dissolved in 0.1 M NaNO3 four times, AH concentration was found to be approximately 780 μg/mL. Molar mass distribution of HA samples was assessed by GPC, both in analytical and semi-preparative scale, using refractive index (RI) and UV/Vis (280 nm) detectors, 0.1 M NaNO3 as mobile phase, flow rate 0.8 mL/min and tracers dextran and pullulan (polymers of similar hydrodynamic volume as compared to HA) as molar mass standards. The samples showed molar mass distributions in the range of 103 to 107 Da, after being precipitated and dissolved four times. For these samples, the average molar mass was found to be in the order of magnitude of 105 Da. In the semi-preparative column Superose, it was possible to separate HA fractions, with molar mass above 105 Da, and free of proteic contaminants, in an elution interval of 10 minutes
publishDate 2007
dc.date.issued.fl_str_mv 2007-03-20
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.uri.fl_str_mv http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2410
url http://www.teses.ufc.br/tde_busca/arquivo.php?codArquivo=2410
dc.language.iso.fl_str_mv por
language por
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv application/pdf
dc.publisher.none.fl_str_mv Universidade Federal do CearÃ
dc.publisher.program.fl_str_mv Programa de PÃs-GraduaÃÃo em Engenharia QuÃmica
dc.publisher.initials.fl_str_mv UFC
dc.publisher.country.fl_str_mv BR
publisher.none.fl_str_mv Universidade Federal do CearÃ
dc.source.none.fl_str_mv reponame:Biblioteca Digital de Teses e Dissertações da UFC
instname:Universidade Federal do Ceará
instacron:UFC
reponame_str Biblioteca Digital de Teses e Dissertações da UFC
collection Biblioteca Digital de Teses e Dissertações da UFC
instname_str Universidade Federal do Ceará
instacron_str UFC
institution UFC
repository.name.fl_str_mv -
repository.mail.fl_str_mv mail@mail.com
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