海洋紫色杆菌β-琼胶酶的分离纯化及性质

海洋紫色杆菌β-琼胶酶的分离纯化及性质 Purification and Characterization of a New β-Agarase from Marine Bacterium Janthinobacterium 时岩玲 SHI Yanling 1 first-author 于文功 YU Wengong 1 路新枝 LU Xinzhi 1 中国海洋大学医药学院 中国海洋大学医药学院 College of Medicine and Pharmaceutics,Ocean University of China College of Medicine and Pharmaceutics,Ocean University of China 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 Betaproteobacteria.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 agarase AgaY was drastically inhibited by adding cations viz Hg2+,Ag+,Zn2+,Cu2+ and Pb2+ in the assay system.Unlike some reported agarases,the activity of agarase AgaY did not increase but decreased when Na+ or Ca2+ was on a higher ion concentration.The catalytic activity of agarase AgaY was not dependent on cations,especially Na+ and Ca2+.And agarase AgaY was not affected by EDTA. 紫色杆菌 β-琼胶酶 分离纯化 新琼寡糖 β-agarase Janthinobacterium purification and characterization neoagarooligosaccharides Q93 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） 2008-04-01 2021-04-01 4