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.
Highlights
Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC
Using chondroitinases AC and C, we have shown that cartilage chondroitin sulfate contains both 4-sulfated and 6-sulfated disaccharide units in variable proportions, depending on the species and age [9,10] and using chondroitinases B and AC we demonstrated that dermatan sulfates of different origins contain glucuronic and iduronic acid in different proportions and at variable positions [11]
These chondroitinases were isolated by agarose gel electrophoresis [6,7] or by column chromatography procedures based on ion exchange [12], gel permeation [8], adsorption on hydroxyapatite [13], or affinity chromatography [14]
Summary
Flavobacterium heparinum is a soil bacterium that produces several mucopolysaccharidases such as heparinase, heparitinases I and II, and chondroitinases AC, B, C and ABC. Flavobacterium heparinum produces four chondroitinases which form unsaturated disaccharides from dermatan and chondroitin sulfates, i.e., chondroitinases AC [5], B [6], C [7] and ABC [8]. We describe improved conditions for bacterial growth and cell disruption, a reproducible scheme for purification of F. heparinum chondroitinases AC and B, as well as a rapid assay for chondroitinase activities based on decrease of metachromatic shift of the color of dimethylmethylene blue.
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Schedule a callMore From: Brazilian journal of medical and biological research = Revista brasileira de pesquisas medicas e biologicas
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