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.
Eu
J
J
Department of Materials Science and Engineering, University of Utah, Salt Lake City, UT 84112-9202, USA.
Andrade
J
J
D000103:Acetyl-CoA Carboxylase / Q000737:chemistry; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry; D003065:Coenzyme A / Q000494:pharmacology; D001517:Coleoptera / Q000201:enzymology; D004229:Dithiothreitol / Q000494:pharmacology; D004795:Enzyme Stability; D004800:Enzymes, Immobilized; D054889:Fatty Acid Synthase, Type II; D066298:In Vitro Techniques; D007700:Kinetics; D008156:Luciferases / Q000737:chemistry* / Q000378:metabolism; D049449:Luminescence; D011108:Polymers / Q000494:pharmacology; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D013501:Surface-Active Agents / Q000494:pharmacology
D002352:Carrier Proteins; D004800:Enzymes, Immobilized; D011108:Polymers; D011993:Recombinant Fusion Proteins; D013501:Surface-Active Agents; D008156:Luciferases; D054889:Fatty Acid Synthase, Type II; D000103:Acetyl-CoA Carboxylase; C079633:biotin carboxyl carrier protein; D003065:Coenzyme A; D004229:Dithiothreitol