Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography

Detalhes bibliográficos
Autor(a) principal: Aguiar, Jair Adriano Kopke [UNIFESP]
Data de Publicação: 1999
Outros Autores: Michelacci, Yara Maria [UNIFESP]
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Repositório Institucional da UNIFESP
Texto Completo: http://dx.doi.org/10.1590/S0100-879X1999000500007
http://repositorio.unifesp.br/handle/11600/771
Resumo: Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.
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spelling Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatographychondroitinasesmucopolysaccharidasesdermatan sulfatechondroitin sulfateFlavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.Universidade Federal de São Paulo (UNIFESP)UNIFESP, EPM, São PauloSciELOAssociação Brasileira de Divulgação CientíficaUniversidade Federal de São Paulo (UNIFESP)Aguiar, Jair Adriano Kopke [UNIFESP]Michelacci, Yara Maria [UNIFESP]2015-06-14T13:24:52Z2015-06-14T13:24:52Z1999-05-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersion545-550application/pdfhttp://dx.doi.org/10.1590/S0100-879X1999000500007Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 5, p. 545-550, 1999.10.1590/S0100-879X1999000500007S0100-879X1999000500007.pdf0100-879XS0100-879X1999000500007http://repositorio.unifesp.br/handle/11600/771WOS:000080489600007engBrazilian Journal of Medical and Biological Researchinfo:eu-repo/semantics/openAccessreponame:Repositório Institucional da UNIFESPinstname:Universidade Federal de São Paulo (UNIFESP)instacron:UNIFESP2024-07-29T16:52:42Zoai:repositorio.unifesp.br/:11600/771Repositório InstitucionalPUBhttp://www.repositorio.unifesp.br/oai/requestbiblioteca.csp@unifesp.bropendoar:34652024-07-29T16:52:42Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)false
dc.title.none.fl_str_mv Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
title Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
spellingShingle Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
Aguiar, Jair Adriano Kopke [UNIFESP]
chondroitinases
mucopolysaccharidases
dermatan sulfate
chondroitin sulfate
title_short Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
title_full Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
title_fullStr Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
title_full_unstemmed Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
title_sort Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
author Aguiar, Jair Adriano Kopke [UNIFESP]
author_facet Aguiar, Jair Adriano Kopke [UNIFESP]
Michelacci, Yara Maria [UNIFESP]
author_role author
author2 Michelacci, Yara Maria [UNIFESP]
author2_role author
dc.contributor.none.fl_str_mv Universidade Federal de São Paulo (UNIFESP)
dc.contributor.author.fl_str_mv Aguiar, Jair Adriano Kopke [UNIFESP]
Michelacci, Yara Maria [UNIFESP]
dc.subject.por.fl_str_mv chondroitinases
mucopolysaccharidases
dermatan sulfate
chondroitin sulfate
topic chondroitinases
mucopolysaccharidases
dermatan sulfate
chondroitin sulfate
description Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. The purpose of the present study was to optimize the preparation of F. heparinum chondroitinases, which are very useful tools for the identification and structural characterization of chondroitin and dermatan sulfates. We observed that during the routine procedure for cell disruption (ultrasound, 100 kHz, 5 min) some of the chondroitinase B activity was lost. Using milder conditions (2 min), most of the chondroitinase B and AC protein was solubilized and the enzyme activities were preserved. Tryptic soy broth without glucose was the best culture medium both for bacterial growth and enzyme induction. Chondroitinases AC and B were separated from each other and also from glucuronidases and sulfatases by hydrophobic interaction chromatography on HP Phenyl-Sepharose. A rapid method for screening of the column fractions was also developed based on the metachromatic shift of the color of dimethylmethylene blue.
publishDate 1999
dc.date.none.fl_str_mv 1999-05-01
2015-06-14T13:24:52Z
2015-06-14T13:24:52Z
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://dx.doi.org/10.1590/S0100-879X1999000500007
Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 5, p. 545-550, 1999.
10.1590/S0100-879X1999000500007
S0100-879X1999000500007.pdf
0100-879X
S0100-879X1999000500007
http://repositorio.unifesp.br/handle/11600/771
WOS:000080489600007
url http://dx.doi.org/10.1590/S0100-879X1999000500007
http://repositorio.unifesp.br/handle/11600/771
identifier_str_mv Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 5, p. 545-550, 1999.
10.1590/S0100-879X1999000500007
S0100-879X1999000500007.pdf
0100-879X
S0100-879X1999000500007
WOS:000080489600007
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv Brazilian Journal of Medical and Biological Research
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv 545-550
application/pdf
dc.publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
publisher.none.fl_str_mv Associação Brasileira de Divulgação Científica
dc.source.none.fl_str_mv reponame:Repositório Institucional da UNIFESP
instname:Universidade Federal de São Paulo (UNIFESP)
instacron:UNIFESP
instname_str Universidade Federal de São Paulo (UNIFESP)
instacron_str UNIFESP
institution UNIFESP
reponame_str Repositório Institucional da UNIFESP
collection Repositório Institucional da UNIFESP
repository.name.fl_str_mv Repositório Institucional da UNIFESP - Universidade Federal de São Paulo (UNIFESP)
repository.mail.fl_str_mv biblioteca.csp@unifesp.br
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