•
(E.C.1.5.1.29) * Pricing updated September 23rd, 2011. Prices are subject to change without notice. Shipping charges are not included. ** One unit of FMN-reductase converts 1.0 µmole of FMN and NADH to FMNH2 and NAD per minute at pH 7.9 at 37°C. Specific Activity: ≥ 2 U/mg protein. For other size, please contact us Click to order or to get further information |
||||||||||
Synonyms: NAD(P)H:flavin oxidoreductase, NAD(P)H:flavin mono-nucleotide oxidoreductase, NAD(P)H(2):FMN oxidoreductase, NAD(P)H-FMN reductase, NAD(P)H-dependent FMN reductase, NAD(P)H:FMN oxidoreductase, riboflavin mononucleotide reductase, flavin mononucleotide reductase
NOVOCIB's bacterial (E. coli) NAD(P)H-dependent FMN-oxidoreductase is a recombinant protein of ca. 26kDa overexpressed in E.coli. The sequence of cloned Fre (SwissProt accession number P0AEN1) was confirmed by DNA sequencing (100% identity).
NAD(P)H:flavin oxidoreductases (or flavin reductases) catalyze the reduction of riboflavin, FMN, and FAD by NADPH and NADH. These enzymes are present in all microorganisms, including luminous marine bacteria, and also in mammals. Flavin reductases have been classified into two groups. The first group includes flavoproteins that use a flavin prosthetic group for the electron transfer from NAD(P)H to the flavin substrate (flavin reductase P from Vibrio harveyi). In the second group, enzymes do not contain any prosthetic group. Fre, NAD(P)H:flavin oxidoreductase from E. coli, belongs to this second group of enzymes.
Applications Coupling of bacterial luciferase to FMN-NAD(P)H oxidoreductase has been used to provide ultrasensitive analytical tools for the quantification of NADH and the substrates of NADH-, NADPH- dependent enzymes (e.g. glucose, lactate, malate, ethanol, sorbitol, oxaloacetate). Although FMN-reductase often present in luciferase enzyme preparations may be sufficient for producing light in the presence of NAD(P)H, highly purified and characterized Fre enzyme can offer some advantages such as an increased sensitivity, better control of the signal intensity and duration, and saving of the luciferase enzyme (see below, Figure 1).
(^Top)
NAD(P)H:flavin oxidoreductases (or flavin reductases) catalyze the reduction of riboflavin, FMN, and FAD by NADPH and NADH. These enzymes are present in all microorganisms, including luminous marine bacteria, and also in mammals. Flavin reductases have been classified into two groups. The first group includes flavoproteins that use a flavin prosthetic group for the electron transfer from NAD(P)H to the flavin substrate (flavin reductase P from Vibrio harveyi). In the second group, enzymes do not contain any prosthetic group. Fre, NAD(P)H:flavin oxidoreductase from E. coli, belongs to this second group of enzymes.
Applications Coupling of bacterial luciferase to FMN-NAD(P)H oxidoreductase has been used to provide ultrasensitive analytical tools for the quantification of NADH and the substrates of NADH-, NADPH- dependent enzymes (e.g. glucose, lactate, malate, ethanol, sorbitol, oxaloacetate). Although FMN-reductase often present in luciferase enzyme preparations may be sufficient for producing light in the presence of NAD(P)H, highly purified and characterized Fre enzyme can offer some advantages such as an increased sensitivity, better control of the signal intensity and duration, and saving of the luciferase enzyme (see below, Figure 1).
 Unit Definition: One unit of FMN-reductase converts 1.0 µmole of FMN and NADH to FMNH2 and NAD per minute at pH 7.9 at 37°C. Assay conditions: 100mM Tris-HCl, pH 7.9, 250µM NADH, 25µM FMN. Specific Activity: ≥ 2 unit/mg protein. Purity: controlled by 10% AA SDS-PAGE. |
Figure 1: Calibration curves (log-log plot) for NADH obtained using purified luciferase (50µg/ml, produced and purified by NovoCIB) in the presence (Red) or absence (Blue) of exogenous FRE (10mU/ml).
 Luminescence signal (5 seconds) was measured immediately after NADH addition (5µL) to 200µL-well containing 0.1M KH2PO4 pH=6.9, 0.02% decanal, 50µM FMN, 2mg/ml BSA. Reaction was followed in a FluoroSkan Ascent FL (ThermoLabsystems) microtiter plate reader. |
|
|
References (with links to PubMed) |
Download this document "NovoCIB's FMN Reductase" 