The Study On Production And Purification Of Laccases Produced By Roseovarius Sp. IOB-7 And Biodegradation Of Pollutants By It

Laccases are polyphenol oxidases ,which can catalyze the oxidation of phenolic and aromatic amine compounds with molecular oxygen as the electron acceptor. Laccases are highly useful biocatalysts for various biotechnological applications because of their wide range of substrates. With the deepening of research , laccases are considered to be some of the most promising enzymes for future industrial applications. Then purification of laccases becomes an advanced research hotspot because it is the key for application. Roseovarius sp.IOB-7 shows the activity of laccases. In present study, the fermentation and purification methods suitable for laccase in Roseovarius sp. IOB-7 are established; and then enzymatic properties and influnences of the factors such as pH, temperature on enzyme activity are examined. In addition,characteristics of IOB-7 laccases for degradating of phenolic and dye are also studied.Results show that: Roseovarius sp. IOB-7 is able to oxidize iodide (I-) to molecular iodine (I2),and forms purple colonies when it is incubated on 2216E agar plates containing iodide and soluble starch. Laccases from IOB - 7 are extracellular laccases, and enzyme activity reaches the maximum after incubation 48 h. The growth of IOB - 7 is promoted with 40 ?mol/L CuSO4, and the secretion of laccases also increases. Moreover enzyme activity is 1.75 times higher than it in control group.Supernatant is purified by satuated (NH4)2SO4 precipitation,dialysis and ion- exchange chromatography, finally enzyme activity of purified laccases is 12 times higher than that of orginal supernatant. The enzyme activity is 602.34 U/mg after directly concentrated by ultrafiltration, which is 77 times higher than the orginals . SDS-PAGE and Native PAGE show that molecular weight of laccase from IOB-7 is about 66 KD.The optimal pH for purified laccases from IOB-7 is 5.5 and they are stable from pH 4.5 to 7.0, the relative activity remain more than 80% after incubating for 5 h . The optimal temperature for them is 50? and they are also stable at 30? to 70?, the relative activity remain more than 50%after incubating at 60 ? for 30min. The Km and Vmax value of laccases from IOB-7 toward substrate KI are 2.983 mmol/L and 1.945?mol.L-l.min-1 under the condition of pH 5.5 and 20 ?.Laccases from IOB-7 can slightly catalyze the degradation of phenol, 4 -chlorophenol , 2, 4 - chlorophenol , and adding ABTS can promote the degradation of phenolic substances. The ability of laccases from IOB-7 to removal Congo red is poor,while it shows potential to malachite green. Under the condition of alkaline , adding 10 U/L laccases from IOB-7 ,the degradation rate of malachite green increases 77.3% when the pH is 7, while 88.3% for pH 9.