文献类型：期刊文献

中文题名：AOT/异辛烷反胶束萃取纯化胰蛋白酶及酶变性失活问题的解决

英文题名：Purification of Trypsin by Using AOT/Isooctane Reverse Micellar Extraction and Overcoming Denaturation and Precipitation in the Process

作者：陈霖杰[1];曹学君[1]

机构：[1]华东理工大学生物工程系生物反应器工程国家重点实验室,上海200237

年份：2007

卷号：27

期号：1

起止页码：75

中文期刊名：中国生物工程杂志

外文期刊名：China Biotechnology

收录：CSTPCD;;北大核心:【北大核心2004】；CSCD:【CSCD2011_2012】；

语种：中文

中文关键词：反胶束;胰蛋白酶;乙醇

外文关键词：Reversed miceUar Trypsin Ethanol

摘要：用反胶束技术分离纯化蛋白质,具有高选择性、易于大规模操作等优点,具有良好的工业应用前景。但是离子型表面活性剂形成的反胶束体系萃取蛋白质容易引起蛋白质的变性,这是由于离子型表面活性剂的强电荷作用所导致的。对用AOT/异辛烷反胶束体系从胰酶粗提物中萃取胰蛋白酶进行了研究,通过在反胶束相加入乙醇,解决了反胶束萃取蛋白质时蛋白质变性失活的问题。并且由于乙醇的加入大大减少了分相的时间,简化了实验步骤,优化了实验方法,使此技术在工业上的大规模应用成为可能。通过优化各种实验条件,胰蛋白酶的前萃取率达到90%,反萃取率接近100%。最终得率为88%。纯化后的比活提高了5倍多,从300U/mg左右提高到了1800U/mg。
Trypsin was purified from crude material of pig pancreas by AOT/isooetane reversed micellar system. The influence of the main operating parameters such as ethanol concentration in forward and backward extraction, pH, KCl and AOT concentration, temperature were investigated. Forward and backward extraction recovery of trypsin reached almost 90% and neared 100%, respectively. Finally, about 88% of total yield was obtained, and the specific activity of trypsin was increased to over 1800U/mg with purification factor of 5 folds more. In AOT-isooctane reverse micellar extraction system, denaturation or precipitation of proteins always occured due to strong electrostatic interaction between AOT-proteins molecules. It had been resolved by adding ethanol into reverse micellar system, and no denaturation was observed. Otherwise, the phase separation time was shortened significantly because of ethanol added. It was only 10 minutes or less to reach phases separation after forward and backward extraction. If this method can he applied in industry, efficiency will he greatly improved.