The Purification And Application Of FMN:NADH Oxidoreductase From Photobacterium Phosphoreum

Luminescent bacteria p. phosphoreum DB was screened as a strain of highFMN:NADH Oxidoreductase activity. Thus, FMN:NADH Oxidoreductase of P.phosphoreum DB was purified by ultrasonication, osmotic shock method,chromatographic separation methods, which provided convenience for the furtherstudy of the couple enzymes（luciferase and FMN:NADH Oxidoreductase）. Thisresearch also focused on the application of couple enzymes system: The optimumcondition of P. phosphoreum DB couple enzymes bioluminescence in vitro wasinvestigated; The optimum mixing proportion of couple enzymes from P. leiognathiYL and P. phosphoreum DB was determined, under which condition couple enzymessystem in vitro could achieve maximum luminescent intensity. All of these provided anew idea to improve the detection sensitivity in NADH by couple enzymes luminoussystem in vitro.1. P. phosphoreum DB was selected from seven strains of luminescent bacteria(DB,h1, h2, h5, Hw1, YL, XB) to produce FMN:NADH Oxidoreductase. The crudeenzyme was extracted from the seven strains of luminescent bacteria respectivelythrough the same steps of ultrasonication, ammonium sulfate salting-out andSephadexG-25desalination. Parallel tests showed that crude enzyme from P.phosphoreum DB was of higher FMN:NADH Oxidoreductase activity than othercrude enzymes extracted from the other six luminescent bacteria, so P. phosphoreumDB is the best source of FMN:NADH Oxidoreductase. DB belongs to P. phosphoreum,which can emit gentle blue-green light (maximum emission wavelength at473nm)and sustain for10h.2. FMN:NADH Oxidoreductase of P. phosphoreum DB was purified.FMN:NADH Oxidoreductase was released from cells by ultrasonication or osmoticshock method and purified through DEAE Sepharose CL-6B and Sephacryl S-100.Results showed both ultrasonication and osmotic shock method could achieve purification. FMN:NADH Oxidoreductase was identified prelimily as monomer andthe molecular weight of28kD by SDS-PAGE.3. The optimum condition of couple enzymes bioluminescent system in vitro fromP. phosphoreum DB was established. For maximum luminous intensity, the properamount of substrates was as follows: NADH（0.01mmol/L）400μL, FMN-Na2（10mmol/L）1.5μL, dodecyl aldehyde（27mmol/L）125μL. The optimum mixingproportion of couple enzymes from P. leiognathi YL and P. phosphoreum DB wasdetermined. When the concentration of substrate NADH was0.1mmol/L, theoptimum mixing proportion of couple enzymes extracted from P. leiognathi YL and P.phosphoreum DB respectively was1:1; When the concentration of substrate NADHwas0.01mmol/L, the optimum mixing proportion of couple enzymes extracted from P.leiognathi YL and P. phosphoreum DB respectively was3:2; Under the optimummixing proportion of couple enzymes, the couple enzymes bioluminescent system invitro could achieve highest luminous intensity.