摘要
对由原核载体表达的人乙醛脱氢酶2(Aldehyde dehydrogenase 2,简称ALDH2)纯化工艺、酶活性改善、稳定性以及保存条件分别进行了参数的优化,以期为ALDH2商品化剂型开发提供理论依据。通过NAD(P)+酶活测定法,检测不同纯化工艺、金属离子以及不同保存条件对ALDH2的酶活性的影响。通过SDS-PAGE检测ALDH2在模拟胃液和胰液中的稳定性。结果显示,低离子磷酸盐缓冲液透析有利于ALDH2酶活性的恢复,且真空冷冻干燥处理可导致ALDH2酶活性下降。K+、Zn2+、Mg2+、Mn2+、Ca2+均能提高ALDH2酶活性。ALDH2在模拟胃液中稳定性良好,但在模拟胰液中迅速被降解。与此同时,ALDH2酶液在-20℃下能良好地保持其稳定性及酶活,但在4℃和30℃下保存一个月酶活急剧下降。以上结果表明,低离子磷酸盐缓冲液透析法、K+均能提高ALDH2的酶活性,同时该酶可耐受模拟胃液的降解作用,且添加山梨酸钾的ALDH2于-20℃可良好地保持其稳定性及酶活。
In this article,the purification process,enzyme activity improvement,stability and storage conditions of ALDH2 expressed by prokaryotic vector were investigated,expecting to give a reference to the development of commercial dosage form of ALDH2.By NAD(P)+enzyme activity test,the influences of different purification processes,some metal ions and varied storage conditions on the enzyme activity of ALDH2 were respectively studied.By SDSPAGE,the stability of ALDH2 in simulated gastric fluid and simulated pancreatic juice was researched.Results indicated that lower salt solution dialysis was helpful to the recovery of the enzyme activity of ALDH2 and vacuum freezedrying could lead to the loss of enzyme activity.K+,Zn2+,Mg2+,Mn2+and Ca2+could all improve the enzyme activity.ALDH2 could keep stable in simulated gastric fluid,but it quickly degraded in simulated pancreatic juice.At the same time,ALDH2 could keep stable and active when stored at-20 ℃,but the enzyme activity of ALDH2 quickly decreased when stored at 4 ℃ and 30 ℃for a month.Based on above results,it is obvious that both lower salt solution dialysis and K+can improve the ALDH2 enzyme activity.And ALDH2 can stand the degradation of simulated gastric fluid.ALDH2 added with potassium sorbate can be stored under-20℃and show perfect stability and enzyme activity.
出处
《氨基酸和生物资源》
CAS
2015年第4期61-66,共6页
Amino Acids & Biotic Resources
基金
国家自然科学基金项目(81172178)
湖北省国际科技合作重点计划项目(2006CA013)
湖北省科技攻关计划项目(2007AA201C27
2007AA301B24)
