Purification and Characterization of Chondroitinase ABC from Proteus vulgaris , an Iraqi Clinically Isolate

Forty-eight isolates were found belonging to Proteus sp. with isolation percentage of 38 out of 125 urine samples which were collected from urinary tract infected patients. Proteus vulgaris and Proteus mirabilis represent 18.7% (9 isolates), and 81.25% (39 isolates) respectively. The production of chondroitinase enzyme was studied in a medium which contained chondroitin sulphate as substrate. One hundred per cent of P. vulgaris showed the ability to produce chondroitinase. P.v 8 isolate produced the highest enzymatic activity which reached about 150 units/ml. Chondroitinase was extracted and purified by precipitation with 60% saturation of ammonium sulphate, dialysis, and followed by column chromatography on Sephadex 6B. The specific activity increased to 5000 (units/mg), with 35.2% yield and 17.4 fold purification. Optimum pH for activity and stability was 8.0; the optimal temperature for activity was 37°C, but the stability of chondroitinase was maintained 100% at 20–40°C for 30 min. Chondroitinase activity increased to 120% and 113% when the enzyme was incubated with Mg⁺² and Ca⁺² respectively, while NH₄Cl and KCl reduced the activity to 95% and 86% respectively. The human cartilage was degradable by the purified enzyme after incubation for 14 days at 37°C. Direct injection of chondroitinase at 0.1 mg/ml in knee cartilage of mice, showed changes in tissues. It may be concluded that chondroitinase enzyme may work as a virulence factor by catalysing the hydrolysis of chondroitin sulphate of cartilage and increasing the tissue permeability to invade the cartilage tissue by P. vulgaris which causes destruction of cartilage and inflammation in humans.