地衣芽孢杆菌2709蛋白酶分离纯化研究

Separation and Purification of Protease from Bacillus licheniformis 2709

对产自地衣芽孢杆菌(B. licheniformis)2709的碱性蛋白酶通过乙醇沉淀、盐析、DEAE阴离子交换层析、凝胶层析4步纯化,最终获得电泳纯的酶。以去酰胺度及酶比活力为指标,对碱性蛋白酶分离纯化条件进行优化。结果发现:提纯酶的比活力达61069U/mg,纯化倍数为38.7,活性回收率为19.3%,去酰胺度为20.9%。并研究该酶的基本酶学特性,结果发现:该酶最适作用pH值为10.0,最适反应温度为50℃。40℃保温2h后该酶保持80%以上的活力,在pH8～11之间有较高的pH值稳定性。

A procedure comprising alcohol precipitation,ammonium sulfate precipitation,DEAE ion exchange chromatography and gel filtration chromatography was used to prepare electrophoretically pure alkaline protease from the fermentation supernatant of Bacillus licheniformis 2709.Deamidation degree and alkaline protease activity were evaluated to optimize the procedure.The results indicated that the activity of purified protease was 61069 U/mg;final purification factor was 38.7;recovery rate of activity was 19.3%;and deamidation rate was 20.9%.The enzymological properties of alkaline protease were also studied.The optimal reaction conditions for alkaline protease were pH 10.0 and 50 ℃.After incubation at 40 ℃ for 2 h,the protease remained more than 80% activity and was relatively stable at pH 8-11.