星点设计-效应面法优化复合酶解酪蛋白制备抗凝血肽的工艺研究  

Optimization of preparation technology of anticoagulant peptides by complex enzymatic hydrolysis of casein by central composite design-response surface method

作　　者：吴雄 夏建开 苗艳丽 陈绍红[2] 张兆霞 郭婉婷 李棂灵 WU Xiong;XIA Jiankai;MIAO Yanli;CHEN Shaohong;ZHANG Zhaoxia;GUO Wanting;LI Lingling(College of Chemistry and Environment of Guangdong Ocean University,Zhanjiang 524088,China;College of Food Science and Technology,Modern Biochemistry Experimental Center,Guangdong Ocean University,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学化学与环境学院,广东湛江524088 [2]广东海洋大学食品科技学院现代生物化学实验中心,广东湛江524088

出　　处：《药学研究》2019年第6期336-342,共7页Journal of Pharmaceutical Research

基　　金：广东省教育厅2015年重点平台及科研项目(No.2015KTSCX057);广东海洋大学2017年度大学生创新创业训练计划项目(No.CXXL2017140)

摘　　要：目的本文利用星点设计-效应面法优化复合酶解酪蛋白制备抗凝血肽的工艺条件,并测定其体外抗凝血活性与溶栓效果。方法首先运用星点设计-效应面法来确定菠萝蛋白酶、胰蛋白酶、胰糜蛋白酶和木瓜蛋白酶这4种酶各自酶解酪蛋白的最佳反应条件,然后在各酶的最佳反应条件下对酪蛋白进行复合酶解,制得酪蛋白复合酶解液,再运用凝血酶滴定改进法来测定其体外抗凝血活性,并用抗凝血活性最高的酪蛋白复合酶解液进行体外溶栓实验。结果4种酶酶解酪蛋白的最佳反应条件分别如下:菠萝蛋白酶:pH为6.50、温度为60.00℃、酶用量为12000.00U/(g底物)、时间为3.00h、料水比为1∶7(m/V);胰蛋白酶和胰糜蛋白酶均为:pH为8.00、温度为50.00℃、酶用量为6000.00U/(g底物)、时间为3.00h、料水比为1∶7(m/V);木瓜蛋白酶:pH为6.50、温度为60.00℃、酶用量为4000.00U/(g底物)、时间为3.00h、料水比为1∶7(m/V)。结论本实验条件下,加酶组合为“菠萝蛋白酶-胰蛋白酶-胰糜蛋白酶-木瓜蛋白酶”的复合酶解制备的酪蛋白酶解液具有最高的体外抗凝血活性,为100.0ATU·mL^-1;新西兰大白兔的血栓线用100.0ATU·mL^-1的酶解液浸泡24h后全部溶解,且兔血与100.0ATU·mL^-1酪蛋白酶解液的比例(V/V)为1∶1和4∶6的样品组放置48h仍不凝固。ObjectiveUsing the central composite design-response surface method to optimize the preparation technology of anticoagulant peptides by complex enzymatic hydrolysis of casein,and its anticoagulant activity and thrombolytic effect in vitro were determined.MethodsFirst,the central composite design-response surface method was used to determine the optimal reaction conditions for the hydrolysis of casein by four enzymes bromelain,trypsin,pancreatic chymotrypsin,and papain.Then,the casein complex enzymatic hydrolysis was obtained by complex enzymatic hydrolysis of casein under the optimal reaction conditions of each enzyme.The thrombin titration modification method was used to determine the anticoagulant activity in vitro and the casein complex enzymatic hydrolysate with the highest anticoagulant activity was used for in vitro thrombolytic experiments.ResultsThe optimal reaction conditions of the four enzymes for hydrolyzing casein were as follow,bromelain:pH was 6.50,temperature was 60.00℃,enzyme dosage was 12 000.00 U·g^-1,time was 3.00 h,material to water ratio was 1∶7(m/V);trypsin and pancreatic chymotrypsin:pH was 8.00,temperature was 50.00℃,enzyme dosage was 6 000.00 U·g^-1,time was 3.00 h,material to water ratio was 1∶7(m/V);papain:pH was 6.50,temperature was 60.00℃,enzyme dosage was 4 000.00 U·g^-1,time was 3.00 h,material to water ratio was 1∶7(m/V).ConclusionUnder the experimental condition,the casein enzymolysis solution obtained by the combination of the four enzymes-bromelain,trypsin,pancreatic chymotrypsin,and papain-had the highest anticoagulant activity(100.0 ATU·mL^-1);The casein enzymolysis solution with highest anticoagulant activity can completely dissolve the rabbit thrombus line after soaking for 24 h.And when New Zealand white rabbit blood and the casein enzymolysis solution ratio(V/V)was 1∶1 and 4∶6,the blood still did not coagulate after 48 h.

关 键 词：星点设计-效应面法 酪蛋白 复合酶解 抗凝血肽 体外溶栓 

分 类 号：TQ460.6[化学工程—制药化工]