Specific immobilization of in vivo biotinylated bacterial luciferase and FMN:NAD(P)H oxidoreductase.
Bacterial bioluminescence, catalyzed by FMN:NAD(P)H oxidoreductase and luciferase, has been used as an analytical tool for quantitating the substrates of NAD(P)H-dependent enzymes. The development of inexpensive and sensitive biosensors based on bacterial bioluminescence would benefit from a method to immobilize the oxidoreductase and luciferase with high specific activity. Toward this end, oxidoreductase and luciferase were fused with a segment of biotin carboxy carrier protein and produced in Escherichia coli. The in vivo biotinylated luciferase and oxidoreductase were immobilized on avidin-conjugated agarose beads with little loss of activity. Coimmobilized enzymes had eight times higher bioluminescence activity than the free enzymes at low enzyme concentration and high NADH concentration. In addition, the immobilized enzymes were more stable than the free enzymes. This immobilization method is also useful to control enzyme orientation, which could increase the efficiency of sequentially operating enzymes like the oxidoreductase-luciferase system.
D000103:Acetyl-CoA Carboxylase / Q000235:genetics; D019921:Biotinylation; D002352:Carrier Proteins / Q000235:genetics; D002384:Catalysis; D004800:Enzymes, Immobilized; D004926:Escherichia coli; D038181:FMN Reductase; D054889:Fatty Acid Synthase, Type II; D007700:Kinetics; D008156:Luciferases / Q000235:genetics / Q000378:metabolism*; D008163:Luminescent Measurements; D009247:NADH, NADPH Oxidoreductases / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism