摘要
研究不同条件对地衣芽孢杆菌De株产生胞外蛋白酶的量及其酶活性的影响,结果表明在pH为7.4—8.2范围内,温度为30℃时,培养8—12h的菌株所分泌胞外产物中的蛋白酶活性最高。实验先以半透膜法收集芽孢杆菌的胞外产物,然后再经过硫酸铵沉淀过夜S、ephadex G-100凝胶层析和DEAE-Cellulose离子交换层析及聚丙烯酰胺凝胶电泳等四个步骤的分离纯化后,可以得到含有3种主要蛋白质(BLP1、BLP2、BLP3)成分的胞外蛋白酶,其分子量分别为66.2KD、31.0KD及约20.1KD,所得纯化蛋白酶的蛋白浓度为0.773μg/mL,蛋白回收率为11.66%。实验还发现,纯化的胞外蛋白酶在100℃下作用30min,仍可保持其活力,可见具有相当的热稳定性,而其酶活最佳的pH和温度条件分别为7.8和45—65℃。酶活抑制实验显示EDTA、铜、钴、镁离子等均可成为其酶活抑制因子;而丝氨酸蛋白酶抑制剂甲基磺酰氟(PMSF)、铁、锰、钡、钙离子等对酶活性没有明显影响;锌则会令之酶活性其部分丧失。
The research of Bacillus as a kind of probiotic for aquatic animals is increasing with the demand for environmentfriendly aquaculture, like biocontrol when the treatment is antagonistic to pathogens or bioremediation when water quality is improved. The Bacillus licheniformis srain De isolated from white shrimp（ Litopanaeus vannamie）cuhural ponds can produce many kinds of extracelluar enzyme, moreover, the activity of extracelluar protease is very high. Therefor, the study was carried out to collect it＇s extracellular protease and the physicochemical characters of the enzyme were investigated. The extracelhdar protease of the strain De was collected followed the semiperable membrane method and was purified followed a four-step purification procedure,including ammounium sulfate precipitation, Sephadex G-100 gel filtration chromatography, anion-exchange chromatography on DEAE-cellulose and polyacrylamide gel electrophoresis. The activities of extracelluar protease was tested by the Azocasein digestion method. As results showed that extracellular protease secreted by strain De obtained a higher enzymatic activity when it was cultivated 12-18h at 30℃ and pH 7.4-8.2. Three predominant protein of the extracellular products BLP1 ,BLP2, BLP3 were obtained,whose molecular mass measured by polyacrylamide gel electrophoresis respectively were 66.2KD 31.0KD and rough 20.1KD. The protein content of purified extracellular protease was 0. 773μg/mL, comparatively, its＇purified protein recovery rate was 11.66%. Moreover, the optimum pH and temperature of the extracellular protease was found to he 7.8 and 45-- 65℃. The extracellular protease is thermophilic, since full biological activity is retained after heating at 100℃ for 30min. Enzyme testing for inhibition of the extracellular protease indicated that only EDTA, Cu^2＋ , Co^2＋ and Mg^2＋ can inhihite enzymatic activity, but PMSF, Fe^3＋ , Mn^2＋ , Ba^2＋ , Ca^2＋ can not, and Zn^2＋ can inhihite enzymatic activity to some extent.
出处
《水生生物学报》
CAS
CSCD
北大核心
2006年第3期262-268,共7页
Acta Hydrobiologica Sinica
基金
广东省重大科技专项(A3050304)资助
关键词
地衣芽孢杆菌
胞外产物
纯化
特性分析
Bacillus licheniformis
Extracellular proteases
Purification
Characterization
