高等学校化学学报

高等学校化学学报 ›› 2000, Vol. 21 ›› Issue (5): 702.

• 论文 • 上一篇    下一篇

复合纤维素膜固定化胰蛋白酶反应器及其应用于蛋白质酶解

姜泓海, 邹汉法, 汪海林, 倪坚毅, 张强   

  1. 中国科学院大连化学物理研究所国家色谱研究分析中心, 大连 116011
  • 收稿日期:1999-10-10 出版日期:2000-05-24 发布日期:2000-05-24
  • 通讯作者: 邹汉法(1961年出生),男,博士,研究员,博士生导师,主要从事高效液相色谱、膜色谱、生物色谱、毛细管电色谱和基体辅助激光解吸电离飞行时间质谱研究.
  • 基金资助:

    国家自然科学重点基金(批准号:29635010);国家杰出青年基金(批准号:29725512)

Covalent Immobilization of Trypsin on Glycidyl Methacrylate-Modified Cellulose Membrane as Enzyme Reactor

JIANG Hong-Hai, ZOU Han-Fa, WANG Hai-Lin, NI Jian-Yi, ZHANG Qiang   

  1. National Chromatographic R & A Center, Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116011, China
  • Received:1999-10-10 Online:2000-05-24 Published:2000-05-24

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被引次数

67

摘要： 合成了甲基丙烯酸缩水甘油酯-纤维素复合膜,并以此膜为基质共价键合固定化胰蛋白酶,以N-苯甲酰-L-精氨酰乙酯(BAEE)为底物,应用高效液相色谱系统测定了酶固定化膜柱的催化反应特性。研究结果表明:温度、pH值、离子强度、有机溶剂及蛋白变性剂等都对固定化酶的活力有一定的影响。在最适条件下,固定化胰蛋白酶的活力为17800U/g干膜,蛋白载量为3.6mg/g(≈0.15μmol/g)干膜,活性回收率达到52%.固定化酶表现出较高的使用和储藏稳定性,在40℃下,水解BAEE底物24h活力无显着变化。固定化酶膜柱在4℃冷藏保存100d仍保存90%以上的水解活力。固定化酶反应器被应用于蛋白质酶解的肽谱实验。

关键词: 复合纤维素膜, 固定化胰蛋白酶, 生物反应器, 蛋白质降解, MALDI-TOF-MS

Abstract: The membrane of the activated glycidyl methacrylate (GMA)-modified cellulose was prepared and packed into a column piece by piece to make a microreactor by immobilization of trypsin. The microreactor based on the membrane medium showed the advantages of being cheaper, mechanically strong and chemically stable. The activity of immobilized trypsin towards N-benzoyl-L-arginine ethyl ester(BAEE) was 17800 U/g dry membrane, and was 52 % of that for free enzyme. Besides, the effects of pHvalue of buffer, temperature, ionic strength, organic modifier, and protein denaturants on the activity of immobilized trypsin were investigated in comparison to the free trypsin, and thermal stability also was found especially to be improved after immobilization. The activity of the immobilized trypsin showed no decay after continuously pumping BAEEthrough immobilized trypsin microreactor for 24 h at 40 ℃ and 0.5 mL/min. Finally, the cytochrome Cwas digested by microreactor and the products were analysed by MALDI-TOF-MS. The peptide mapping for a protein by combination of the immobilized enzyme membrane microreactor and MALDI-TOF-MShas been developed.

Key words: Glycidyl methacrylate (GMA)-modified cellulose membrane, Immobilized trypsin enzyme, Biological reactor, Degradation of protein, MALDI-TOF-MS, Cytochrome C

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