Cai Su-Lan, Yan Hao-Lin, He Han-Zhou
The School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, China.
Sheng Wu Gong Cheng Xue Bao. 2004 Jul;20(4):584-9.
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.
多年来，软骨素酶一直是研究糖胺聚糖结构、功能和分布的重要工具。最近，有报道称该酶是一种潜在的化学溶核酶，化学溶核是一种已确立的椎间盘突出症治疗方法。本文采用硫酸铵沉淀、QAE-葡聚糖A50离子交换色谱和葡聚糖G-150凝胶过滤的简单纯化程序，从温和气单胞菌YH311的培养上清液中纯化出一种软骨素酶。还研究了以海藻酸钠或纤维素为载体对纯化后的软骨素酶进行固定化。从温和气单胞菌YH311获得的软骨素酶纯化了55倍，纯度达到95.3%，纯化酶的比活性为31.86u/mg，产率为37%。通过SDS-PAGE测定，温和气单胞菌YH311的软骨素酶分子量为80kD，与金色节杆菌、液化气单胞菌和肝素黄杆菌的软骨素酶AC几乎相同。但其等电点为4.3 - 4.6，远低于微生物软骨素酶AC。在固定于海藻酸钠或纤维素后，软骨素酶的性质发生了很大变化。游离酶的最适温度和pH分别为50℃和7.0，在80℃热处理20分钟后约保留10%的活性，在4℃储存两周后保留47%的活性。固定于海藻酸钠的软骨素酶最适温度和pH分别为40℃和7.0，在80℃热处理120分钟后约保留50%的活性，在4℃储存30天后保留50%的活性。固定于纤维素的软骨素酶最适温度和pH分别为70℃和6.0，在80℃热处理后和4℃储存30天后保留超过70%的活性。固定化产率非常低，海藻酸钠为18.56% ,纤维素为18.86%。
