猪胰腺羧肽酶B提取工艺中的激活条件与稳定性研究  

Studies on the activation condition and stability of pork pancreas carboxypeptidase B in the process of extraction and purification

作　　者：王福清 李素霞[2] 郑勇锋[2] 

机构地区：[1]中国生化制药工业协会,北京100801 [2]华东理工大学生物反应器工程国家重点实验室,上海200237

出　　处：《中国生化药物杂志》2007年第4期250-253,共4页Chinese Journal of Biochemical Pharmaceutics

摘　　要：目的研究猪胰腺羧肽酶B提取工艺中的激活条件以及激活后粗酶液的稳定性,并对酶进行纯化。方法胰腺匀浆、激活,硫酸铵盐析进行初步提取,采用疏水色谱、离子交换色谱和分子筛等方法进行纯化,对胰腺匀浆后的粗酶激活条件进行了研究,考察了不同保存条件下粗酶液的稳定性。结果纯化后的羧肽酶B经SDS-PAGE电泳鉴定为电泳纯。比活为152 u/mg,纯化过程的活性总回收率为51%。粗酶液中羧肽酶B的激活受各种条件影响,特别是激活温度和激活时间,4℃下激活80 h,或37℃下,激活6.5 h,均可获得最大酶活,激活后的粗酶液可直接于35%(NH4)2SO4中保存,保存60 h的酶活损失小于5%。结论通过调整激活温度与激活时间可以获得猪胰腺羧肽酶B粗酶液最大的激活效果,激活后的粗酶液可于35%(NH4)2SO4中短期保存。Purpose The activation condition of carboxypeptidase B （CPB）after pork pancreas tissue homogenation and the stability of CPB were studied in the process of purification, and CPB was further purified after activation. Methods The process to extract and purify CPB from pork pancreas was as follows：fresh or frozen pork pancreas tissue was homogenated, activated and salted out with ammonium sulfate, then purified by hydrophobic chromatography, ion-exchange chromatography and molecular sieve chromatography. Specially, the activation condition was studied thoroughly, including different pH buffers, different activation temperatures and different durations of activation. The stability of crude CPB was studied under different storing conditions. Results Special activity of purified CPB was 152 u/mg protein and the total activity recovery rate was 51%. The activity of CPB in tissue homogenation was mainly influenced by temperature and duration of activation. The maximum activity of CPB could be obtained to activate the crude extraction at 4℃ for 80 h or at 37℃ for 6.5 h. Less than 5% of total activity of CPB was lost while kept in 35% （NH4）2SO4 for 60 h after activation. Conclusion The highest activity of crude CPB can be obtained through adjusting the activated temperature and time and the crude activated CPB could be kept in 35 % （NH4）2SO4 temporarily.

关 键 词：羧肽酶B 羧肽酶原B 酶原激活 稳定性 

分 类 号：TQ464.53[化学工程—制药化工]