摘要
将磁性可再生性固定化酶应用于微波辅助蛋白酶解。包覆了Si O2的Fe3O4磁性粒子(Fe3O4@m Si O2)表面经3-(2,3-环氧丙氧)丙基三甲氧基硅烷偶联剂(GLYMO)与亚氨基二乙酸(IDA)反应的产物(GLYMO-IDA)修饰,加入Cu2+形成金属螯合配体后利用Cu2+与木瓜蛋白酶作用固定木瓜蛋白酶。在优化条件下,获得的固定化酶酶活为1 158.8 U,酶活保留率达44.1%。所获得的固定化木瓜蛋白酶具有易再生、再生后酶活稳定的特点。再生5次后酶活平均值为1 014 U,再生率达88%。HPLC分析表明,微波辅助结合固定化可再生酶可用于黑蚂蚁蛋白的快速酶解,获得的色谱图轮廓与自由酶酶解结果相似。酶解过程快,多肽丰富且易与底物分离,表明可再生固定化酶结合微波辅助有助于蛋白的酶解与多肽的制备。
The magnetic regenerated immobilized papain is applied for microwave assisted enzymatic digestion （MAED）.Fe3O4 magnetic nanoparticles were coated with SiO2by sol-gel method and Fe3O4@ mSiO2 were formed and then modified by the reaction products of 3-Glycidyloxy propyl trimethoxy silane coupling agent （GLYMO）and iminodiacetic acid （IDA）.After Cu2＋was chelated on the Fe3 O4@mSiO2-GLYMO-IDA,the papain was immobilized on.In optimized conditions,the activity of the immobilized papain was 1 158.8 U and the rate of remained activity of papain was 44.1%.After five times of regeneration,the average activity of the immobilized papain was 1 014 U and regeneration rate was about 88%.Coupled with MAED,the immobilized papain was applied for black ants protein digestion.The HPLC analysis shows that during 30 min,the digestion product from immobilized papain MAED could be easily separated from the substrate and its responding HPLC profiles were similar to the product from free papain digestion,reflecting that the digestion speed of black ants protein with immobilized papain was fast and efficient.When protocol is combined with immobilized enzyme, MAED would be helpful for the protein digestion and potential peptides preparation.
出处
《桂林理工大学学报》
CAS
北大核心
2014年第4期748-754,共7页
Journal of Guilin University of Technology
基金
国家自然科学基金项目(21065003
21265004)
广西自然科学基金项目(2012GXNSFAA053030)
关键词
磁性粒子
固定化酶
木瓜蛋白酶
黑蚂蚁蛋白
magnetic particles
immobilization enzyme
papain
black ant protein
