摘要
目的探索超声波对胰蛋白酶催化的影响效果和作用方式。方法研究胰蛋白酶经不同频率、不同功率、不同时间的超声波处理后酶活力的变化;用动力学参数变化和光谱学探索其影响催化的机理。结果经超声波处理后酶活力普遍升高。用15 kHz5、0 W超声波处理胰蛋白酶3 min,酶活力可提高45.4%。将粗酶用SephadexG75凝胶色谱,分部液在聚丙烯酰胺凝胶电泳检验显示出一条带,说明酶被纯化到电泳纯程度。对纯酶进行动力学分析,结果表明经超声波处理后Km值变小,Vmax值也降低,说明超声波处理使酶对底物的亲和力增大。超声波处理不会改变胰蛋白酶分子的构型,但会改变酶分子的构象。结论超声波对胰蛋白酶催化活性的提高可能是由于改变酶分子的构象、增强酶分子对底物的亲和力的结果。
Purpose This investigation was to explore the effect of ultrasound on catalytic activity of trypsin and its action mechanism. Methods The activity of trypsin treated by ultrasound in different frequency, and different power for different time was studied. The change of kinetics parameters and spectra of trypsin was detected. Results It was indicated that the activity of trypsin treated by ultrasound was higher than that of the enzyme untreated. The activity of the enzyme treated with ultrasound in 15 KHz, 50 W for 3 min increased by 45.4 %. The crude enzyme was purified with Sephadex G75 column. The elution was studied by polyacrylamide gel electrophoresis. One protein strap showed on the electrophoresis gel. It was indicated that the enzyme had been purified to the extent of electrophoresis purity. The result of kinetics analysis showed that both Km and Vmax of the enzyme decreased after ultrasonic treatment. It was indicated that trypsin affinity to its substrate increased after being treated with ultrasound. Further research on ultraviolet absorption spectrum, ultraviolet differential spectrum and fluorescence emission spectrum indicated that the configuration did not change, but the conformation of enzyme molecules changed when the enzyme was treated by ultrasound. Conclusion The activity increase of trypsin treated with ultrasound was probably because that ultrasound treatment changed the conformation of enzyme and raised the affinity of enzyme to substrate molecules.
出处
《中国生化药物杂志》
CAS
CSCD
北大核心
2009年第4期230-234,238,共6页
Chinese Journal of Biochemical Pharmaceutics
基金
国家自然科学基金(10074016)资助
关键词
胰蛋白酶
酶活力
超声波动力学
光谱分析
trypsin
enzyme activity
ultrasonic kinetics analysis
spectrum analysis
