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›› 2008, Vol. 8 ›› Issue (6): 1195-1199.

• 生化工程专栏 • 上一篇    下一篇

高强度耐有机溶剂蛋白酶的纯化及性质

姚忠 孙蓓蓓 李霜 何冰芳   

  1. 南京工业大学制药与生命科学学院 南京工业大学制药与生命科学学院 南京工业大学 制药与生命科学学院 南京工业大学制药与生命科学学院 江苏省生物工业技术重点实验室
  • 收稿日期:2008-03-31 修回日期:2008-12-01 出版日期:2008-12-20 发布日期:2008-12-20
  • 通讯作者: 何冰芳

Purification and Characterization of Protease with High Stability in Organic Solvebts

YAO Zhong Sun Bei-bei LI Shuang HE Bing-fang   

  1. College of Life Science and Pharmacy, Nanjing University of Technology College of Life Science and Pharmaceutical engineering, Nanjing University of Technology College of Life Science and Pharmaceutical Engineering. Nanjing University of Technology College of Life Science and Pharmacy, Nanjing University of Technology, The Provincial Key Laboratory of Industrial Biotechnology
  • Received:2008-03-31 Revised:2008-12-01 Online:2008-12-20 Published:2008-12-20
  • Contact: HE Bing-fang

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摘要： 分离纯化了自行筛选的耐有机溶剂的地衣芽孢杆菌Bacillus licheniformis YP1所产的溶剂稳定性蛋白酶. 发酵液经硫酸铵沉淀、疏水层析及强阳离子交换层析纯化，得到电泳纯的蛋白酶；分子量约为28 kDa，纯化蛋白酶的比酶活达到1.18′105 U/mg，纯化倍数为37.2，酶活回收率为20.8%. 纯化的YP1蛋白酶对50%(j)的多种亲水或疏水有机溶剂具有很高的耐受性，其中50%(j)的DMF和DMSO能显著促进YP1蛋白酶活力. YP1蛋白酶为Zn2+蛋白酶，最适反应温度为55℃，最适反应pH为10.0，pH 8.0~13.0范围内具有高活力. 以酪蛋白为底物，YP1蛋白酶的米氏常数Km为0.048 g/L.

关键词: 碱性蛋白酶, 耐有机溶剂, 地衣芽孢杆菌, 纯化, 表征

Abstract: A novel solvent-tolerant protease was produced by a solvent-tolerant Bacillus licheniformis YP1 strain isolated from soil. The protease was purified by precipitation with ammonium sulfate, hydrophobic interaction chromatography and cation exchange chromatography, leading to 37.2-fold purification with 20.8% recovery rate. The product showed eletrophoretic homogeneity, as identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), and the specific activity of the purified protease reached 1.18×105 U/mg. SDS-PAGE analysis indicated that the relative molecular mass of the protein was about 28 kDa. The protease was stable and active in all the tested solvents and the protease activity was significantly enhanced in the presence of 50%(j) DMSO and DMF. The protease was considered as a Zn2+-complexed enzyme with an optimal reaction temperature of 55℃. The enzyme was stable in the pH range of 8.0~13.0, with an optimum pH of 10.0. The Michaelis constant for caseinolytic activity was 0.048 g/L.

Key words: alkaline protease, solvent tolerance, Bacillus licheniformis YP1, purification, stability