An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases

An improved methodology to produce Flavobacterium heparinum chondroitinases, important instruments for diagnosis of diseases

Author Aguiar, JAK Google Scholar
Lima, C. R. Google Scholar
Berto, AGA Google Scholar
Michelacci, Y. M. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract Chondroitinases are very important tools for the identification and structural analysis of proteoglycans. Enzymic analysis with Flavobacterium heparinum chondroitinases has shown that chondroitin sulphate and dermatan sulphate structures are modified in many human diseases, suggesting a diagnostic value for these enzymes. Furthermore, it was recently shown that F. heparinum chondroitinases AC and B inhibit tumoural cell growth, invasion and angiogenesis. Due to the increasing importance of F. heparinum chondroitinases, we investigated optimized conditions for preparation and assay of chondroitinases AC, B and C. the Dimethylmethylene Blue assay was modified and fully developed to measure the chondroitinase activities of crude extracts of F. heparinum. This method estimates chondroitin sulphate or dermatan sulphate depolymerization upon the digestion of chondroitinase, and was compared with A(232), which measures the unsaturated products formed. Trypticase was the best culture medium, both for bacterial growth and enzyme induction. the chondroitinases were solubilized by ultrasound under conditions that do not completely disrupt the cells, suggesting that they are located at the periplasmic space. Maximum chondroitinase induction occurred in the presence of 0.2-1.0 g/l chondroitin sulphate. Chondroitin sulphate-degraclation products were also inducers, but heparin and heparan sulphate were not. Chondroitinases AC, B and C were separated from each other by hydrophobic-interaction chromatography on Phenyl-Sepharose HP. When contaminant proteins were first removed from crude extract by Q-Sepharose, the chondroitinases could be purified to homogeneity in this phenyl-Sepharose chromatographic step.
Keywords chondroitin sulphate
dermatan sulphate
Language English
Date 2003-04-01
Published in Biotechnology and Applied Biochemistry. Malden: Wiley-Blackwell, v. 37, p. 115-127, 2003.
ISSN 0885-4513 (Sherpa/Romeo, impact factor)
Publisher Wiley-Blackwell
Extent 115-127
Access rights Closed access
Type Article
Web of Science ID WOS:000181820500003

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