Properties of firefly luciferase immobilized through a biotin carboxyl carrier protein domain.

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 degrees C and 25 degrees C for 22 h. Incubation at 37 degrees C 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 micromol/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 2001 John Wiley & Sons, Ltd.