Biochimica et Biophysica Acta 923 (1987) 291-301 291 Elsevier BBA22491 Isolation and characterization of an induced chondroitinase ABC from Flavobacteriumheparinum Yfira M. Michelacci, Denise S.P.Q. Horton and Carlos A. Poblaci6n Escola Paulista de Medicina, Departamento de Bioqulmica, S~o Paulo (Brazil) (Received 31 July 1986) (Revised manuscript received 5 November 1986) Key words: Chondroitinase ABC; Induced enzyme; (Flaoobacterium) During the investigation of alternative methods for the large scale preparation of chondroitinases AC, B and C from Flavobacterium heparinum, a new chondroitinase activity was observed. This new enzyme, like the other chondroitinases, acts as an eliminase, forming unsaturated sulfated disaccharides from dermatan and chondroitin sulfates. In contrast to the chondroitinases previously described, which are endoglycosidases, this chondroitinase ABC cleaves the glycosidic linkages in an exolytic fashion, beginning at the reducing end of the substrate molecules. The oligosaccharides formed as transient products by the action of either chondroitinases or testicular hyaluronidase upon dermatan and chondroitin sulfates are also rapidly degraded by the chondroitinase ABC, regardless of their size or the presence of A-4,5 unsaturation in the terminal uronic acid residue. The maximum activity of the chondroitinase ABC occurs at 30°C and at pH 6.0-7.5. Only 15% of the activity was observed at 37°C, indicating that the enzyme is very sensitive to thermal denaturation. It is strongly inhibited by phosphate ions and is also inhibited by the unsaturated disaccharides formed. Introduction Flavobacterium heparinum, a soil bacterium iso- lated by Payza and Korn [1], has been shown to produce several enzymes which degrade glycos- aminoglycans. These include a series of inducible enzymes able to degrade heparin and heparan sulfate to their basic constituents [2-5] and a series of enzymes able to degrade chondroitin sulfates. The latter series of enzymes includes chondroitinases, glycuronidases and sulfatases [6]. The chondroitinases which have been isolated and characterized so far are: a chondroitinase AC that degrades chondroitin 4- and 6-sulfates and hy- Correspondence: Y.M. Michelacci, Departamento de Bioquimica, Escola Paulista de Medicina, Caixa Postal 20372, 04044 $5o Pauio, Brazil. aluronic acid [6]; a chondroitinase B that degrades the iduronic acid-containing regions of dermatan sulfate [7]; and a chondroitinase C that acts upon hyaluronic acid and the non-sulfated or the 6- sulfated regions of the chondroitin sulfate mole- cules [8]. All these enzymes act as eliminases. The elimination reaction results in a A-4,5 site of unsaturation in the uronic acid residue, leaving the chemical structure of the glycosamine reducing end unaltered [9]. The chondroitinases C and AC are constitutive enzymes whereas chondroitinase B is induced when the bacterium is grown in the presence of chondroitin sulfates or dermatan sulfate [7,8]. During the investigation of alternative methods for the large-scale preparation of the chondro- itinases B, C and AC a new enzymatic activity which degrades chondroitin 4- and 6-sulfates and dermatan sulfate was observed. This new 0304-4165/87/$03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)