Preparation and purification of Flavobacterium heparinum chondroitinases AC and B by hydrophobic interaction chromatography
Autor(a) principal: | |
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Data de Publicação: | 1999 |
Outros Autores: | |
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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Repositório Institucional da UNIFESP |
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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 |
_version_ |
1814268309115240448 |