溶栓酶AprE9912D的纯化工艺及酶学性质考察  

Investigation on Purification and Characterization of Thrombolytic Enzyme AprE9912D

作　　者：张秀 张莉[1] 李晓媛 杜仕静 谭正怀[1] ZHANG Xiu;ZHANG Li;LI Xiaoyuan;DU Shijing;TAN Zhenghuai(Sichuan Academy of Chinese Medicine Sciences,Chengdu 610041)

机构地区：[1]四川省中医药科学院,四川成都610041

出　　处：《中国医药工业杂志》2025年第2期198-204,共7页Chinese Journal of Pharmaceuticals

基　　金：四川省省级科研院所基本科研业务费项目(2022JDKY0013)。

摘　　要：溶栓酶AprE9912D(AprE)是由贝莱斯芽孢杆菌9912D分泌的碱性蛋白酶,具有纤溶活性。该研究通过比较可溶蛋白与包涵体2种表达形式的产量、纯化效果及其他参数来确认AprE后续的纯化工艺,并探究了纯化品的体外溶栓作用、溶血性和酶学性质。相较于在可溶蛋白中纯化AprE,包涵体经过洗涤、快速透析复性和强阴离子交换色谱柱纯化,所得的AprE纯度达95%以上,复性率为52.7%,得率为11.2%,且具有较高的酶比活(50 456.7 IU/mg)。因此,宜采用包涵体形式表达AprE。10 000 IU/mL AprE在体外能显著溶解陈旧血栓,溶栓率为16.7%(P<0.001);并且,AprE浓度达到2 000 IU/mL时的溶血率也仅为1.0%,说明其安全性良好。AprE的最适反应温度为35~45 ℃,最适pH值为7.0~8.0。该研究为天然新型溶栓药物AprE的进一步开发提供了科学依据。Thrombolytic enzyme AprE9912D(AprE),an alkaline protease secreted by Bacillus velezensis 9912D,shows fibrinolytic activity.The purification process of AprE was determined by comparing yields,purification levels and other parameters of soluble protein and inclusion body for the following experiment.After purification,thrombolytic effect,hemolytic property and characterization of AprE was explored in vitro.Compared with the purification of AprE from the soluble protein,AprE in inclusion body was obtained through washing,rapid dialysis renaturation,anion-exchange chromatography column with purity of over 95%,renaturation rate of 52.7%,yield of 11.2%,and higher specific enzyme activity(50 456.7 IU/mg).Therefore,it was appropriate to express AprE in the form of inclusion bodies.In vitro,10 000 IU/mL of AprE could significantly dissolve old thrombi,the thrombolysis rate was 16.7%(P<0.001).The hemolysis rate of AprE at a concentrationof 2 000 IU/mL was only 1.0%,indicated good safety.The optimal temperature and pH value for the reaction of AprEwere 35-45 ℃ and 7.0-8.0,respectively.This study provided a scientific basis for the future research of the naturalthrombolytic enzyme AprE.

关 键 词：溶栓酶 碱性蛋白酶 纯化 溶栓 溶血 酶学性质 

分 类 号：TQ461[化学工程—制药化工]