嗜热芽孢杆菌高温蛋白酶HS08的化学修饰研究

分离纯化了嗜热芽孢杆菌产生的耐高温蛋白酶HS08,以SDS-PAGE凝胶电泳和Superdax75预装柱凝胶过滤,测定蛋白酶HS08的分子质量为30.6ku,同时研究了酶专一性抑制剂对酶活性的影响。结果表明,蛋白酶HS08的活性被酶活性中心丝氨酸残基专一性抑制剂PMSF以及金属离子螯合剂EDTA强烈抑制,而NBS、WRK、TNBS、Phenylgloxalhydrate等修饰剂对酶活性无明显抑制作用。K+、Na+、Ca2+、Mg2+、Zn2+等金属离子对酶活性影响实验结果表明,Zn2+可使该酶活性明显提高,Cu2+对该酶活性有抑制作用。因此,该酶属于金属离子激活的丝氨酸族蛋白酶。蛋白酶HS08的酶动力学实验表明,以BSA为底物,PMSF可使蛋白酶HS08最大反应速率Vmax下降50%,而对其米氏常数Km影响不大。

Chemical Modification of a Protease HS08 from Thermophilic Bacillus Strain

The thermo stable protease HS08 secreted from Thermophilic Bacillus strain was isolated and purified.The molecular mass of 30.6ku was estimated by SDS-PAGE and Superdax 75 gel filtration chromatogram.Effects of some protease inhibitors on the protease HS08 were investigated.The results proved that EDTA and PMSF inhibited dramatically the protease activities and the other inhibitors,such as NBS,WRK,TNBS,DTT and phenylgloxal hydrate did not inhibited the protease.The protease activity was activated by Zn 2+ but was inhibited by Cu 2+.So the protease HS08 belonged to metal-activated serine protease.The maximum velocity of the protease was decreased by 50%,but the Michaelis-Menten coefficient Km did not changed with BSA as substrate at 0.075mmol/L PMSF.