[Purification and immobilization of chondroitinase from Aeromonas sobria YH 311]

Chondroitinase has been used as an important tool in the study of the structure, function and distribution of glycosaminoglycans for many years. Recently, the enzyme has been reported to be a potential enzyme for chemonucleolysis, an established treatment for intervertebral disc protrasion. In this paper, a chondroitinase had been purified from the culture supernatant of Aeromonas sobria YH311 using a simple purification procedure of ammonium sulfate precipitation, QAE-Sephadex A50 ion exchange chromatography and Sephadex G-150 gel filtration. The immobilization of purified chondroitinase using sodium alginate or cellulose as carriers has also been studied. The chondroitinase obtained from Aeromonas sobria YH311 was purified 55-fold to 95.3% pure, the specific activity of the purified enzyme was 31.86u/mg and the yield was 37%. The molecular weight of chondroitinase from Aeromonas sobria YH311 was determined by SDS-PAGE to be 80kD, which was almost the same as those chondroitinase AC from Arthrobacter aurescens, Aeromonas liquefaciens and Flavobacterium heparinum. But its isoelectric point was 4.3 - 4.6, which was far lower than the microbial chondroitinase AC. After the immobilization on sodium alginate or cellulose, the properties of chondroitinase changed greatly. The optimum temperature and pH of the free enzyme were 50 degrees C and 7.0 respectively, and about 10% activity remained after heat treatment at 80 degrees C for 20 minutes, and 47% activity remained after two weeks storage at 4 degrees C. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40 degrees C and 7.0 respectively, about 50% activity remained after 80 degrees C heat treatment for 120 minutes and 50% remained after 30 days storage at 4 degrees C. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70 degrees C and 6.0 respectively, and more than 70% activity remained after heat treatment at 80 degrees C and 30 days storage at 4 degrees C. The yield of the immobilization was very low, with 18.56% for alginate and 18.86% for cellulose.