Lavi J T, Raunio R P, Stahlberg T H
Wallac Oy, Turku, Finland.
J Biolumin Chemilumin. 1990 Jul-Sep;5(3):187-92. doi: 10.1002/bio.1170050308.
A modified purification method for bacterial luciferases and NAD(P)H:FMN oxidoreductases is described which uses FMN-Sepharose alone or coupled to DEAE ion exchange chromatography for the simultaneous purification of luciferase and the various oxidoreductases from Vibrio harveyi, a bright mutant of Vibrio harveyi, Vibrio fischeri, and Photobacterium phosphoreum. This purification method is compared with DEAE-Sepharose Cl 6B fractionations from these organisms. Both methods allow the separation of oxidoreductases specific for either NADH or NADPH. The use of FMN-Sepharose coupled to DEAE-Sepharose fractionation allows the isolation of highly purified enzymes. Lacking interfering factors, these are very suitable for various analytical applications based on bacterial bioluminescence enzymes. The partially purified enzymes from the affinity column have higher specific activities than those obtained using DEAE-Sepharose.
本文描述了一种改良的细菌荧光素酶和NAD(P)H:FMN氧化还原酶的纯化方法，该方法单独使用FMN-琼脂糖或与DEAE离子交换色谱联用，用于从哈维弧菌、哈维弧菌的一个明亮突变体、费氏弧菌和磷光发光杆菌中同时纯化荧光素酶和各种氧化还原酶。将该纯化方法与这些生物体的DEAE-琼脂糖Cl 6B分级分离法进行了比较。两种方法都能分离出对NADH或NADPH具有特异性的氧化还原酶。使用与DEAE-琼脂糖分级分离联用的FMN-琼脂糖可分离出高度纯化的酶。由于缺乏干扰因素，这些酶非常适合基于细菌生物发光酶的各种分析应用。来自亲和柱的部分纯化酶比使用DEAE-琼脂糖获得的酶具有更高的比活性。
