| Five luminous bacterial strains were isolated from Qingdao coast and characterized by a combination of 16SrDNA sequences, the morphology observation, physiological test, molecular and biochemical analysis. In comparison with the similar strains reported, the five strains are identified as P.leiognathi, V.harveyi, P.phosphoreum, V.splendidus, V.fischeri.Sensitivity screening examination indicated that P.leiognathi YL was most sensitive to the antibiotics and heavy metals among the five strains. YL strain has a maxium wavelength for emission at 471nm and the strong light lasted for 16h. The optimum growth and luminescence conditions of YL were determined as: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。The medium was composed of 0.5% peptone, 0.1% yeast extraction, 0.01% FePO4, 2-4% NaCl, 0.4% glycerin and seawater.P.leiognathi YL was selected to produce luciferase and FMN:NADH oxidoreductase(the couple enzymes) for its stable product ability. The optimum enzyme-producing conditions were determined : Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min。Luciferase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL luciferase mixture, 53μL FMN-Na(10mM), 100μL Na2S2O4(34mM), 100μLdodecanal(27mM).FMN:NADH oxidoreductase was assayed at room temperature by measuring the light emission upon the rapid injection of 1.0mL oxidoreductase mixture, 53μL FMN-Na(10mM), 200μL NADH(0.14mM), 100μL dodecanal(27mM). The luminescence of the coupled enzyme system was more stable than luciferase system.The couple enzymes were released from the cells by sonicate and purified followed the procedures: precipitated by solid ammonium sulfate; elution on DEAE-Sepharose CL-6B and DEAE-Sephadex columns. The molecular weights of oxidoreductase is 28KDa. Luciferase has two subunits and their molecular weights are 36KDa and 41KDa respectively. No activity was found when either of the isolated subunits is absent. The optimum reaction conditions of the couple enzymes are: Initial pH 7.5;temperature 25-30℃;shaking speed 150r/min. K+,Ca2+,Na+,Mg2+ promoted the activity of the couple enzymes while Fe2+,Zn2+,Sn2+,Hg2+,Ba2+ inhibited the activity. Antibiotics had no effect on the activity of the couple enzymes. The couple enzymes had a good activity at 4℃and the activity decreased with the temperature raised over 40℃.The concentration of chloramphenicol in shrimp and turbot tissues was detected with P.leiognathi YL. The chloramphenicol in the tissues was extracted by ethyl acetate. The optimal test inoculum is 107cells/mL. 5mL chloramphenicol solution was added into 5mL inoculum. The mixture solution reacted for 30min at 27℃with the shaking speed 150r/min. The concentration of chloramphenicol ranging from 0.14 to 1.0μg/L was determined by the luminescence inhibition rate of P.leiognathi YL. The detection limit is 0.14μg/kg. The concentration of heavy metals(Hg2+,Cr6+,Cd2+)in shrimp tissues was also detected with P.leiognathi YL. The ions of heavy metal in the tissues were extracted by cineration. The detection procedure of heavy metals(Hg2+,Cr6+,Cd2+)was the same as the chloramphenicol. The concentration of Hg2+ ranging from 0.007μg/L to 0.28μg/L was detected, the detection limit was 0.007μg/kg.The detection concentration of Cr6+ is 0.9-30.0μg/L and the detection limit was 0.9μg/kg. The detection concentration of Cd2+ was 5.4-65.0μg/L and the detection limit was 5.4μg/kg.Sensitivity screening examination indicated that the couple enzymes were most sensitive to Hg2+and Cr6+. The concentrations of Hg2+and Cr6+ in shrimp tissues were detected with the couple enzymes respectively. The ions of heavy metal in the tissues were extracted by cineration. 500μL ion solution was added into the enzyme mixture to react for 15min at 4℃, and the activity of the couple enzymes was detected. The detection concentration of Hg2+ was 0.0007-0.030μg/L and the detection limit was 0.0007μg/kg. The detection concentration of Cd2+ was 0.05-5.0μg/L and the detection limit was 0.08μg/kg. NADH in E.coli cells was released by sonicate and promoted the luminescence of the couple enzymes. The results provided data to detect the total number of E.coli with the couple enzymes. |
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