嗜热芽孢杆菌蛋白酶HS08的分离纯化研究

从高温土壤中分离得到的一株产耐热中性蛋白酶的嗜热芽孢杆菌,该菌分泌的胞外蛋白酶粗酶液经80%饱和度硫酸铵沉淀、DEAE-Sepharose Fast Flow阴离子交换层析和Superdax75凝胶层析分离纯化后,纯化倍数提高4．36倍,得率为5．3%,经SDS-PAGE电泳测得其分子量为30．6kDa。酶的最适温度与pH实验表明,该酶最适温度为65℃、最适pH为7．5,并且该酶在50℃时表现出1h以上的稳定性。该蛋白酶的活性受到丝氨酸族蛋白酶专一性抑制PMSF和金属离子螯合剂EDTA强烈抑制,Zn2+能提高酶活性,因此该蛋白酶为Zn2+激活的丝氨酸族蛋白酶。

Study on Isolation and Purification of HS08 Protease from Thermophilic Bacillus Strain

A Thermophilic Bacillus strain for secreting thermostable neutral protease was isolated from high temperature soil sample.The purification steps included 80% saturated ammonium sulfate sedimentation first,then with DEAE-Sepharose fast flow anion exchange chromatogram and Superdax 75 gel filtration on AKTA purification 100 protein liquid chromatogram system,and the final purification-fold 4.36 and recovery rate 5.31% were obtained.The molecular mass 30.6kDa was estimated by SDS-PAGE.The optimal pH and the protease temperature were 7.5 and 65℃,respectively.The protease was stable at 50"C during for 1 h incubation.The protease was inhibited markedly by serine protease inhibitor PMSF（phenylmethylsulfonyl fluoride）and metal ion chelator EDTA（ethylene diamine tetraacetic acid）.Andalso the protease activily is enhanced by Zn2+.So the protease belongs to Zn2+activated serine protease.