摘要
通过硫酸铵沉淀、QAE SephadexA5 0柱层析及SephadexG 1 5 0凝胶过滤等纯化步骤 ,对源自温和气单孢菌YH31 1的ChSase进行了分离纯化。结果表明 ,ChSase经上述纯化步骤后被纯化了 5 5倍 ,其最终纯度可达 95 %以上 ,比活为 31 86u mg。经SDS PAGE及IFE测定可知该酶的分子量约为 80kD ,等电点为 4 3～ 4 8。将纯化后的ChSase用海藻酸钠或纤维素固定化后 ,ChSase的热稳定性及贮存稳定性均可得到大幅度的提高 :固定化酶用 80℃水浴处理 1 2 0min或于 4℃冰箱放置 30d后仍可保留 5 0 %以上的相对活力 ;但固定化酶的收率较低 ,仅为 1 8 5 6 %和1 8 86 %。
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℃ and 7 0 respectively, and about 10% activity remained after heat treatment at 80℃ for 20 minutes, and 47% activity remained after two weeks storage at 4℃. The chondroitinase immobilized on sodium alginate had the optimum temperature and pH of 40℃ and 7 0 respectively, about 50% activity remained after 80℃ heat treatment for 120 minutes and 50% remained after 30 days storage at 4℃. The chondroitinase immobilized on cellulose had the optimum temperature and pH of 70℃ and 6 0 respectively, and more than 70% activity remained after heat treatment at 80℃ and 30 days storage at 4℃. The yield of the immobilization was very low, with 18 56% for algina
出处
《生物工程学报》
CAS
CSCD
北大核心
2004年第4期584-589,共6页
Chinese Journal of Biotechnology
