一种碱性蛋白酶在高压CO_2-水-乙醇体系中的稳定性

Study on Stability of Alkaline Proteinase in the Carbon Dioxide-Water-Ethanol System at Elevated Pressure

基于超(近)临界流体抗溶剂沉淀法分离与纯化蛋白质的发展,酶以及蛋白质的稳定性是一个关键因素.本工作以碱性蛋白酶粗品(由地衣芽孢杆菌2709#发酵生产)为目标蛋白,考察了其在不同CO2压力的作用下,在水-乙醇溶液中的稳定性情况,并进一步探索了酶活降低的原因,寻找保持酶活的办法.实验中所用CO2压力为0~7MPa,温度为35℃,乙醇浓度为0%~20%(wt).实验结果表明,在一定的实验研究范围内,杂蛋白沉淀出来的同时,碱性蛋白酶可以保持80%以上的相对酶活.实验结果为超(近)临界CO2沉淀法分离与纯化碱性蛋白酶提供了技术保障.

In the precipitation seperation of protein with compressed pressure CO2, the protein stability is a key factor. To test its stability, alkaline proteinase (Subtilisin, from Bacillus licheniformis 2709#) was chosen as a model protein in this process and the proteinase activity and hydrolysis were investigated in detail. In this precipitation process, the ethanol was used as a precipitation assistant in aqueous solution of crude alkaline proteinase. The effects of some operating parameters, including CO2 pressure, ethanol concentration, initial pH of solution and processing time etc, on the loss of enzyme activity were measured. The study was performed with pressure range 0~7MPa, temperature 35℃, concentrations of ethanol 0%~20%(wt) and initial pH of solution 5~8. It was found that the operating parameters were very important for retaining alkaline proteinase with high activity. The results indicate that the activity of alkaline proteinase could remain up to 80% at suitable operation conditions while the protein impurities precipitation is achieved. It was shown that this precipitant CO2 is likely can be used in separating and isolating alkaline proteinase.