摘要
短小芽孢杆菌产生的碱性蛋白酶BP经CM—Sephadex-C-50和Sephadex-G75两个柱层析,得到了聚丙烯酰胺凝胶电泳纯的酶组分,比活力从1307μ/mg提高到5538μ/mg.活力回收为21%,酶水解酪蛋白的最适反应温度为50℃,最适pH为9.5,Mn^2+、Ca^2+对酶有激活作用,Hg2+、Ag^+对酶有抑制作用.酶的热稳定性不高,但在Ca^2+保护下,热稳定性明显提高.酶的最适作用底物为酪蛋白,对血红蛋白、蛇毒蛋白、牛血清蛋白、卵蛋白、核糖核酸酶也有水解作用.对酪蛋白的Km为0.62%,V_max为50μg/min.DFP可完全抑制酶活性,PMSF和NBS也严重抑制酶活力,PCMB、_o-PTH和EDTA几乎不抑制酶活力.纯酶的分子量为25000Dal.该酶蛋白含有17种氨基酸,其中甘氨酸(Gly)和丙氨酸(Ala)为主要氨基酸.
Protease BP from B. pumilus was purified to homogenety judged by polyacrylamide gel electrophoresis. The purification procedure included ion-encharge chromatography on CM-Sephadex-C-50 and Sephadex-G-75 chromatography. The enzyme was purified 4. 26-fold with a yield of 21%. The molecular weight of the enzyme was determined by SDS-PAGE to be about 25000 Dal. The enzyme was the most active at 55 C and pH 9. 5. The enzyme activity was stimulated by Mn2+ and Ca2+, but was inhibited by Ag+ and Hg2+. The enzyme was unstable at 50 ℃-60℃. However, in the present of Ca2+ , the stability increased so that more than 80% of the original activity was maintained on heating at 50℃ for 60 min and 60% at 55℃ for 60 min. The optimum substrate for the enzyme was casein. The enzyme could effect on haemoglobin, snake venom protein, bovine serum albumin etc. , but its effect on lysozyme, protamine sulfate and r-globuline was poor. The Km value for casein was 0. 62% and Vmax was 50 μg/min. The enzyme activity was inhibited completely by DEP and was inhibited by PMSF and NBSF too. PCMB, o-PTH and EDTA showed little inhibitory effect on the enzyme. The protease includes 17 amino acids, among which, Gly and Ala are dominant amino acids.
出处
《微生物学报》
CAS
CSCD
北大核心
1994年第4期293-300,共8页
Acta Microbiologica Sinica
关键词
短小芽孢杆菌
蛋白酶
提纯
Bacillus pumilus, Protease, Purification and property
