鲎血蓝蛋白的分离纯化及其酚氧化酶活性的初步研究  

Separation and Purification of Hemocyanin from Limulus and Its Phenoloxidase Activity

作　　者：刘永豪 张干欢 董鸿睿 康信煌 张国光 吴育廉 邓春梅 LIU Yonghao;ZHANG Ganhuan;DONG Hongrui;KANG Xinhuang;ZHANG Guoguang;WU Yulian;DENG Chunmei(College of Chemistry and Environment,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

出　　处：《化学与生物工程》2022年第6期44-49,共6页Chemistry & Bioengineering

基　　金：广东省大学生创新创业训练计划项目(S201910566105);广东海洋大学创新强校项目(Q17092);湛江市海洋生物材料研究重点实验室项目(2015A06004);湛江市海洋资源化学综合利用研究重点实验室项目(2014A06007)。

摘　　要：采用饱和硫酸铵盐析法、透析除盐和丙烯葡聚糖凝胶柱层析分离纯化鲎血蓝蛋白,通过紫外可见吸收光谱和SDS-PAGE图谱对其进行定性分析,再通过单因素实验和响应面实验研究了温度、pH值、邻苯二酚与鲎血蓝蛋白浓度比、时间等因素对鲎血蓝蛋白酚氧化酶活性的影响。结果表明,纯化后的鲎血蓝蛋白在280 nm、340 nm处有特征吸收峰,有2个蛋白亚基,分子量约为66 kDa,为典型的节肢动物血蓝蛋白;在温度为36.7℃、pH值为6.8、邻苯二酚与鲎血蓝蛋白浓度比为1.7∶1、时间为2.1 min的最佳条件下,鲎血蓝蛋白酚氧化酶活性最高,为83.3 U·min^(-1)·mL^(-1)。We separated and purified the hemocyanin from Limulus by saturated ammonium sulfate salting-out method,dialysis desalting method,and allyl dextran gel column chromatograph;meanwhile,we qualitatively analyzed the purified hemocyanin by UV-Vis absorption spectrum and SDS-PAGE profile.Moreover,we studied the effects of temperature,pH value,concentration ratio of 1,2-benzenediol to hemocyanin from Limulus,and time on the phenoloxidase activity of hemocyanin from Limulus by single-factor experiments and response surface methodologies.The results show that the purified hemocyanin from Limulus have characteristic absorption peaks at 280 nm and 340 nm,and have two protein subunits with the molecular weight of about 66 kDa,which is a typical arthropod hemocyanin.The purified hemocyanin shows the highest phenoloxidase activity of 83.3 U·min^(-1)·mL^(-1) under the conditions as follows:the temperature is 36.7℃,the pH value is 6.8,the concentration ratio of 1,2-benzenediol to hemocyanin from Limulus is 1.7∶1,and the time is 2.1 min.

关 键 词：鲎 血蓝蛋白 分离纯化 酚氧化酶活性 

分 类 号：O629.73[理学—有机化学]