| White Rot Fungi are defined by the ability to transfer wood into white spongy materials. This kind of fungi can completely degrade lignin to CO2 and H2 O in pure culture. Laccase is an important and valuable ligninolytic enzyme with great potential in the biotechnological applications. It is mainly produced by white rot fungi and often secreted extracellularly as different isoenzymes. Ganoderma sp.En3 is a valuable fungal strain isolated by our laboratory, which can degrade various synthetic dyes efficiently. The degradation ability of Ganoderma sp.En3 is closely related to laccase. In this study, we purified three laccase isoenzymes secreted by Ganoderma sp.En3 and evaluated the differences among these isoenzymes on their biochemical properties. Furthermore, we tested the ability of these laccase isoenzymes to degrade different types of dyes and polycyclic aromatic hydrocarbons(PAHs) with different structures. The main results of our research are described as follows:Firstly, we optimized the composition of medium for high production of laccase via Plackett-Burman design and Response-Surface method. The yield of laccase was efficiently promoted and the optimum condition was 46.468g/L glucose, 6.490g/L yeast extract and 3.572 mM CuSO4 as inducer. After the optimization, the highest laccase activity was increased to 11.69U/m L, which was 47.23% higher than the initial laccase activity.In GYP medium, after the addition of copper ion, four laccase isoenzymes were detected and named as En3-Lac-1, En3-Lac-2, En3-Lac-3 and En3-Lac-4. En3-Lac-2, En3-Lac-3 and En3-Lac-4 were successfully separated and purified by alternate use of ion-exchange and hydrophobic chromatography after fractionated acetone precipitation. The specific activities of each isoenzyme are 329.03, 382.73 and 192.35U/mg, respectively. And the total recovery was 22.94%, mainly En3-Lac-4(19.73%). The homogeneity of the purified laccase isoenzymes was confirmed by SDS-PAGE and Native-PAGE. En3-Lac-2, En3-Lac-3 and En3-Lac-4 were monomeric proteins with molecular weights of about 74, 72 and 56 kDa, respectively. The study of biochemical characteristics of each isoenzyme revealed that the three enzymes had similar optimum pH and temperature when ABTS was used as substrate. ABTS was the best substrate tested for En3-Lac-2 and En3-Lac-3, but En3-Lac-4 showed the highest affinity to DMP. En3-Lac-2 showed stronger pH stability and thermostability. En3-Lac-2 also had a stronger ability to tolerate metal ions and organic solvents compared with other two isoenzymes.Different laccase isoenzymes purified from Ganoderma sp.En3 were used to degrade different types of synthetic dyes. The degradation ability of these laccase isoenzymes was different. There existed distinct substrate specificity among these isoenzymes. Different laccase isoenzymes had different decolorization capabilities. En3-Lac-2 had a stronger capability for decolorizing the triphenylmethane and indigo dye, while En3-Lac-4 had a stronger ability to decolorize the azo and anthraquinone dye. En3-Lac-3 showed the weakest decolorization ability, which could only degrade MG. The kinetic study on the decolorization ability of each isoenzyme indicated that there existed a distinct difference on the substrate specificity among these enzymes. En3-Lac-2 catalyzed the decolorization of IC with the highest efficiency(Kcat=4.03 s-1), while MG for En3-Lac-3(Kcat=2.58 s-1) and RBBR for En3-Lac-4(Kcat=1.26 s-1). En3-Lac-4 had the largest range of substrates, which could efficiently degrade all the dyes except AF and RB5. In order to promote the decolorization efficiency of laccases, the effect of different mediators on the degradation of AF by En3-Lac-4 was studied and the decolorization efficiency was increased 118-fold by the mediator syringaldehyde. The differences in decolorization capability of laccase isoenzymes were eliminated after adding the mediator syringaldehyde.Among the three laccase isoenzymes purified in our research, En3-Lac-3 had the weakest degradation ability, but there existed a positive synergistic effect of different laccase isoenzymes on the decolorization capability. Combination of En3-Lac-3 with other two isoenzymes greatly promoted the decolorization of different azo dyes. The synergistic effect was related to the rate of different isoenzymes, and the addition of En3-Lac-3 could promote the degradation of azo dyes by En3-Lac-4 more effectively.Furthermore, we evaluated the degradation of different PAHs by these laccase isoenzymes. En3-Lac-3 and En3-Lac-4 could efficiently degrade all the PAHs selected in our research, more specifically, Anthracene(Ant), Fluoranthene(Fla), Fluorene(Flu), Phenanthren(Phe) and Pyrene(Pyr), with a degradation efficiency higher than 85% in 24 h. However, the degradation ability of En3-Lac-2 was relatively weak. It could barely degrade Pyr(less than 10% in 24h). The degradation reactions of PAHs by all the three enzymes were first order reacions. By comparing the rate constant(K0) of each reaction, the degradation ability of PAHs by En3-Lac-2 is Fla>Flu>Phe>Ant>Pyr, and Fla>Phe>Pyr>Ant>Flu for En3-Lac-3 and En3-Lac-4.To sum up, our study suggested that there existed differences among the three laccase isoenzymes purified from Ganoderma sp.En3 on the characteristics and degradation ability. There existed a positive synergistic effect of different laccase isoenzymes on the decolorization of azo dye. This study will contribute to the deep understanding of functions of different laccase isoenzymes and better application of white rot fungi and laccase in the biodegradation of environmental pollutants. |
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