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

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

Author Aguiar, Jair Adriano Kopke Autor UNIFESP Google Scholar
Michelacci, Yara Maria Autor UNIFESP Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract 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.
Keywords chondroitinases
mucopolysaccharidases
dermatan sulfate
chondroitin sulfate
Language English
Date 1999-05-01
Published in Brazilian Journal of Medical and Biological Research. Associação Brasileira de Divulgação Científica, v. 32, n. 5, p. 545-550, 1999.
ISSN 0100-879X (Sherpa/Romeo, impact factor)
Publisher Associação Brasileira de Divulgação Científica
Extent 545-550
Origin http://dx.doi.org/10.1590/S0100-879X1999000500007
Access rights Open access Open Access
Type Article
Web of Science ID WOS:000080489600007
SciELO ID S0100-879X1999000500007 (statistics in SciELO)
URI http://repositorio.unifesp.br/handle/11600/771

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