明亮发光杆菌荧光素酶提取和分离方法优化  

Optimization of the Extraction and Isolation of Luciferase from Photobacterium Phosphoreum

作　　者：阳绮思 张晓晴 杨毅红[1] Yang Qisi;Zhang Xiaoqing;Yang Yihong(UESTC,Zhongshan Institute,Zhongshan 528400;The University of Manchester,Manchester M139PL;Zhongshan Senior Technical Institute,Zhongshan 528400,China)

机构地区：[1]电子科技大学中山学院,广东中山528400 [2]曼彻斯特大学,英国曼彻斯特M139PL [3]中山市技师学院,广东中山528400

出　　处：《广东化工》2021年第20期27-28,11,共3页Guangdong Chemical Industry

基　　金：广东省教育厅普通高校特色创新项目(2019KTSCX215);校级质量工程项目(hhkc201007,ZXKC202005);电子科技大学中山学院教师科研启动项目(415YKQ06)。

摘　　要：荧光素酶在毒性检测、ATP快速检测、报告基因分析等领域有广泛的应用[4]。本研究采用易培养、繁殖快的发光细菌为对象,通过探究明亮发光杆菌菌液最适宜的超声破碎次数;用不同缓冲溶液配制的底物在发光体系中对发光强度与酶活的影响;对单酶发光体系混合底物用量进行梯度比对,优化荧光素酶在发光体系的发光效果;从而建立并优化了从明亮发光杆菌中提取、分离荧光素酶的方法。研究结果表明:在20℃条件下培养16 h得到的明亮发光杆菌菌液,经过超声破碎40次(工作时间15 s,间歇时间30 s,工作效率300 W),此时的发光杆菌菌液的发光强度接近10万RLU,且酶活数值稳定较高,为破碎后菌液的最佳研究状态,同时,细胞的破碎次数过多或过少都会导致酶活偏低;Tris-HCl缓冲溶液比PBS缓冲溶液更有利于荧光素酶在发光体系中发生反应;单酶发光体系混合用量对荧光素酶单酶发光体系的反应影响很大。Photobacterium phosphoreum has a wide range of applications in toxicity detection,ATP rapid detection and reporter gene technique.With the characters of fast growing and easy culture,the study chose Photobacterium phosphoreumas as study object to figure out the optimum ultrasonic breaking times;the effect of substrates which were prepared with different dosage of buffer solutions on luminescent intensity and enzyme activity in Luciferase Bioluminescent system in vitro;the gradient comparison of mixed substrates dosage and the optimization of luciferase luminous effect in single Luciferase Bioluminescent system in vitro;then establish and optimize the method of extracting and isolating luciferase from photobacterium phosphoreum.The results illustrate that Photobacterium phosphoreumas incubates for 16 h at 20℃then ultrasonic breaking for 40 times(working time 15 s,internal time 30 s,working power 300 W),the bacteria solution’s luminescent intensity is close to hundred thousand RLU and with a high enzyme activity value.Meanwhile,if the ultrasonic breaking times are too much or too little,it would cause a low enzyme activity value;Tris-HCl buffer solution is easier than PBS buffer solution for luciferase to react;The amount of mixed substrates in single Luciferase Bioluminescent system also plays an important role in bioluminescent system’s reaction.

关 键 词：明亮发光杆菌 荧光素酶 提取 分离 方法优化 

分 类 号：TQ[化学工程]