Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain
Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain
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DOI：
10.1002/bio.612
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发表时间：
2001-01-01
期刊：
影响因子：
2.9
通讯作者：
Andrade, J
中科院分区：
文献类型：
--
作者：
Eu, JY;Andrade, J
以大肠杆菌生物素羧基载体蛋白（BCCP）结构域和pyralis Photinus萤光素酶（FL）为载体，通过生物素-亲和素相互作用在6%交联琼脂糖珠上固定化了一种融合蛋白。研究了固定化BCCP-FL的几种性质。固定化酶和游离酶的热稳定性差异不显著；在4℃和25℃条件下孵育22小时后，两者均保持至少91%的活性。在37℃条件下孵育22小时，活性明显丧失。测定游离酶和固定化酶的k - m和k（cat）值。游离酶和固定化酶的K-M值相近；而固定化BCCP-FL的k（cat）为游离酶k（cat）的三分之一。294 μ mol/L辅酶A （CoA）和44 mmol/L二硫苏糖醇（DTT）提高了总生物发光量。Triton X-100, Tween 20。peg8000、PVP 40000和PVP 360000对固定化BCCP-FL的生物发光反应没有增强作用。版权所有John Wiley & Sons， Ltd。
A fusion protein, consisting of biotin carboxyl carrier protein (BCCP) domain from Escherichia coli and firefly luciferase (FL) from Photinus pyralis, was immobilized through the biotin-avidin interaction on 6%, cross-linked agarose beads. Several properties of the immobilized BCCP-FL were studied. Immobilized and free enzymes showed no significant difference in thermal stability; both retained at least 91% activity after incubation at 4 degreesC and 25 degreesC for 22 h. Incubation at 37 degreesC for 22 h caused significant activity loss. K-M and k(cat) values were determined for both free and immobilized enzymes. K-M values were similar between free and immobilized enzymes; however, k(cat) of immobilized BCCP-FL, was one-third of the k(cat) of the free enzyme. 294 mu mol/L Co-enzyme A (CoA) and 44 mmol/L dithiothreitol (DTT) enhanced the total bioluminescence output. Triton X-100, Tween 20. PEG 8,000, PVP 40,000 and PVP 360,000 did not enhance the bioluminescence reaction of immobilized BCCP-FL. Copyright (C) 2001 John Wiley & Sons, Ltd.