Purification and characterization of arylsulfatase from Sphingomonas sp AS6330
Purification and characterization of arylsulfatase from Sphingomonas sp AS6330
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DOI：
10.1007/s00253-003-1463-8
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发表时间：
2004-05-01
影响因子：
5
通讯作者：
Kim, HR
中科院分区：
文献类型：
--
作者：
Kim, JH;Byun, DS;Kim, HR
从鞘氨醇单胞菌AS 6330中分离纯化了芳基硫酸酯酶。相对于细胞匀浆，纯度增加了12，800倍，产率约为19.1%。该酶是一种单体蛋白，其表观分子量为62 kDa，通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，和41 kDa，通过凝胶过滤测定。该酶对琼脂和对硝基苯酚硫酸酯的最适反应条件为pH7.0和45 ℃，比活力分别为3.93和97.2U。该酶对琼脂的活性高于其他硫酸化海洋多糖如卟啉、岩藻依聚糖和卡拉胶。该酶水解α-淀粉酶的Km和Vmax分别为54.9 μ M和113 mM/min。200 g琼脂与100 U芳基硫酸酯酶在45 ℃下反应8 h，凝胶强度增加2.44倍，琼脂中97.7%的硫酸酯被水解。
Arylsulfatase was purified from Sphingomonas sp. AS6330 through ionic exchange, hydrophobic- and gel-chromatographies. The purity increased 12,800-fold with approximately 19.1% yield against cell homogenate. The enzyme was a monomeric protein with apparent molecular weight of 62 kDa as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis, and 41 kDa as determined by gel filtration. The enzyme had optimum reaction conditions for hydrolysis of sulfate ester bonds in agar and p-nitrophenyl sulfate (NPS) at pH 7.0 and 45degreesC, with a specific activity of 3.93 and 97.2 U, respectively. The enzyme showed higher activity towards agar than other sulfated marine polysaccharides such as porphyran, fucoidan and carrageenan. The K-m and V-max of the enzyme for hydrolysis of NPS were 54.9 muM and 113 mM/min, respectively. With reaction of 200 g agar with 100 U arylsulfatase for 8 h at 45degreesC, gel strength increased 2.44-fold, and 97.7% of the sulfate in the agar was hydrolyzed.