NOVOCIB's bacterial luciferase is purified from a Photobacterium phosphoreum strain isolated from squid by our team and selected for its brightest luminescence. The luxab gene was amplified by PCR and cloned. The sequences of cloned α and β subunits have shown 94% and 92% identity to P24113 and P12744 proteins of Photobacterium phosphoreum (SwissProt Entry).

Applications: In luminescent marine photobacteria, the production of light results from two successive reactions. The first one is catalyzed by the NAD(P)H-FMN oxidoreductase (EC 1.6.8.1), that produces FMNH₂ acting as a substrate for the second reaction, which is catalyzed by a luciferase (EC 1.14.14.3) to generate light in the presence of an aliphatic aldehyde and molecular oxygen. In the presence of limiting concentrations of NADH substrate, light intensity is proportional to NAD(P)H concentration. The coupling of bacterial luciferase to FMN-NAD(P)H oxidoreductase has been used to provide ultrasensitive analytical tools for the quantification of NAD(P)H and the substrates of NADH-, NADPH- dependent enzymes (e.g. glucose, lactate, malate, ethanol, sorbitol, oxaloacetate)⁽¹⁾.

NOVOCIB's Highly Pure Bacterial Luciferase can be used for NAD(P)H quantification or in dehydrogenase-coupled assays.

Activity: > 500,000 (5.105) RLU per second per �g of protein in the presence of 10�M NADH and 3.5mU/ml FMN-reductase (ref. E-Nov-8) as measured with an Optocomp 1 (Celsis) luminometer. Purity: controlled by 10% AA SDS-PAGE.

Figure 1: Calibration curves (log-log plot) for NADH obtained using Highly Pure Bacterial Luciferase (50�g/ml, NovoCIB, E-Nov 10). Assay condition: Luminescence signal (15-seconds) was measured after NADH addition (5�L) to 200�L-well containing 0.1M KH2PO4 pH=6.9, 0.02% dodecanal, 50�M FMN, 2mg/ml BSA. Reaction was followed in an Optocomp 1 (Celsis) luminometer.

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References (with links to PubMed) 1. P.R. Coulet, L.J. Blum, S.M. Gautier (1989): Optically-based chemical and biochemical sensors for the detection of some drugs and biological compounds J. Pharm. Biomed. Anal. 7(12), 1361-1376