摘要
琼胶酶（agarase）是一类能够降解琼脂糖的糖苷水解酶，在保健食品、医药、化妆品等行业极具应用价值。本研究旨在纯化大肠杆菌BL21（DE3）plySs表达的以包涵体状态存在的重组琼胶酶rAga0917，并对纯化的包涵体蛋白的复性条件进行优化，以获得大量高活性的琼胶酶蛋白。用单因素实验，每次以单一复性条件为变量，用透析复性法探索了初始蛋白浓度、透析时间、温度、非离子去垢剂Triton X-100、甘油等对重组琼胶酶rAga0917包涵体复性的影响。实验结果显示，通过Ni^2＋柱亲和层析，获得了纯度95％以上的蛋白。纯化蛋白浓度低于65μg／mL时，复性蛋白的酶活性与初始蛋白浓度成正比；4℃复性的蛋白，其琼胶酶活性明显高于25℃；随着透析时间的增长，复性效果越来越好；实验还同时发现复性液中的甘油能有效地辅助重组琼胶酶rAga0917复性。
Agarase, a kind of glycoside hydrolase （GH） that hydrolyzes agarose, has potentially broad applications in the health-food, pharmaceutical, and cosmetic industries. The purpose of this research was to purify the recombinant agarase rAga0917 from inclusion body expressed in Escherichia coli BL21 （DE3） plySs and optimize the renaturation conditions of the purified agarose, to obtain a largeamount of agarase with high enzyme activity. By single factor experiments, the effects of protein concentration, dialysis time, temperature, detergent Triton X-100 and glycerol on renaturation were studied. The results revealed that a main protein band （purity〉95%） corresponding to the estimated size of 6xhistine tagged fusion protein was detected afferbeing purified on a Ni^2＋ affinity column. When the initial protein concentration was lower than 65 μg/mL, the agarase activity of renatured protein improved as the protein concentration increasing. Theagarase activityof protein dialyzed at 4 ℃ was more active than that at 25 ℃. As the dialysis time went on, the agarase activity improved. In addition, glycerol might help renaturation of the recombinant agarase rAga0917
作者
郭帅
陈梦仟
朱新术
周晨妍
王燕
Guo Shuai, Chen Mengqian ,Zhu Xinshu ,Zhou Chenyan ,Wang Yan(Synthetic Biology Remaking Engineering and Application Laboratory, College of Life Science and Technology, Xinxiang Medical University, Xinxiang, 45300)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2018年第3期1197-1202,共6页
Genomics and Applied Biology
基金
河南省科技计划项目(152102210127)
新乡医学院科研培育基金(2014QN131)共同资助
关键词
琼胶酶
包涵体
纯化
复性
酶活力
Agarase, Inclusion body, Purification, Renaturation, Enzyme activity
作者简介
通讯作者:王燕,wangyan19820527@163.com
