摘要
从青岛近海海水中分离得到一株高产琼胶酶的海洋紫色杆菌SY12.紫色杆菌SY12的发酵液上清经硫酸铵沉淀,凝胶过滤层析,离子交换层析和凝胶过滤层析等蛋白纯化步骤,得到纯化的野生琼胶酶AgaY.琼胶酶纯化了133.63倍,酶比活力为320.7 U/mg;纯化的琼胶酶经SDS-PAGE检测,显示为单一条带,其相对分子质量约为50×103.研究结果表明琼胶酶AgaY降解琼脂糖的β-1,4-糖苷键,其主要产物为新琼四糖和新琼二糖.对AgaY进行酶学性质研究,发现其最适反应pH为7.0,最适反应温度为40℃;Hg2+,Ag+,Zn2+,Cu2+和Pb2+等金属离子可以显著的抑制琼胶酶AgaY的活力.研究发现,AgaY的催化活性不依赖于离子的存在,AgaY对EDTA不敏感.
An agar-degrading bacterium isolated from the coastal water of Qingdao, China, was identified as a novel species of the genus Janthinobacterium. The genus Janthinobacterium belongs to the class Betaproteobacteria. And it was the first report of the agar-degrading bacterium belonging to Betapro- teobacteria. An extracellular agarase AgaY was purified 133.63-fold by ammonium sulfate precipitation, gel filtration Sephadex G-25, ion-exchange chromatography on Sepharose Q FF and gel filtration Superdex 75, with a specific activity of 320.7 U/mg. The purified enzyme AgaY appeared to be homogeneous under the inspection of SDS-PAGE, and it had a molecular mass of about 50 kDa. AgaY hydrolyzed the β-1,4- linkages of agarose, yielding neoagarotetraose and neoagarohexaose as the main final products. The optimum reaction temperature and pH for agarase AgaY was 40 ℃ and pH 7.0, respectively. The agarase AgaY was very stable below 30 C, and was stable at a wide extent of pH 5.0-9.0. And the activity of aga- rase AgaY was drastically inhibited by adding cations viz Hg^2＋ , Ag^＋ , Zn^2＋ , Cu^2＋ and Pb^2＋ in the assay system. Unlike some reported agarases, the activity of agarase AgaY did not increase but decreased when Na^＋ or Ca^2＋ was on a higher ion concentration. The catalytic activity of agarase AgaY was not dependent on cations, especially Na^＋ and Ca^2＋. And agarase AgaY was not affected by EDTA.
出处
《武汉大学学报（理学版）》
CAS
CSCD
北大核心
2008年第4期497-502,共6页
Journal of Wuhan University:Natural Science Edition
基金
国家重点基础研究发展计划(973)项目(2003CB716402)
国家高技术研究发展计划(863)项目(2004AA625020)
关键词
紫色杆菌
Β-琼胶酶
分离纯化
新琼寡糖
β-agarase
Janthinobacterium
purification and characterization
neoagarooligosaccharides
