Study On Preparation Of Fungal Laccase, Its Catalytic Polymerization Characteristics And Laccase Genes' Cloning

Laccases (benzenediol: oxygen oxidoreductase EC.11.0.32) is a type of multicopper-containing polyphenol oxidases, It has high redox potential and will be capable of oxidizing a wide range of polyphenols. It has various potential applications in several areas such as environmental protection, pulp and paper industry, food processing, organic synthesis and other fields. The main purpose of this research is efficient production of laccase. Some systematic study and analysis have been carried out about the characteristics of the obtained laccase. The expanded culture of Trametes hirsuta BYBF, laccase purification and characterization, applications of laccase in the chlorophenol wastewater have also been investigated.The gene of this laccase from Trametes hirsuta BYBF was also cloned.Fluidized-bed bioreactor has the advantages of simple structure, high oxygen transfer efficiency, high starting speed, small shear force, and so on. The use of mycelial pellets in the fluidized bed for laccase could make mycelial pellets, the fermentation liquid and oxygen contact fully, and so the process of laccase from the white rot fungi could be promoted greatly. A three-phase fluidized-bed bioreactor is used to produce laccase from Trametes hirsuta BYBF. The results show that the mycelium pellets is stable in the fluidized bed and get a better fluidization. Under the optimal growing condition of Trametes hirsuta BYBF with 10%inoculum with 0.3L/min aeration rate, The enzyme activity can achieve 3.36IU/mL after 5 days fermentation. The white rot fungi were was also cultivated with repeating batch fermentation.As compared with the shaker fermentation, the fluidized-bed bioreactor not only increase the enzyme activity,but also greatly shorten the fermentation cycle time and improve the production efficiency. This work lay the foundation for the industrial production of laccase.The laccase of the white rot fungus-Trametes hirsuta BYBF was collected by by centrifugation, filtration, and then purified by ammonium sulfate precipitation, DEAE-Sepharose anion chromatography and Sephadex G-100 gel filtration. After purification with about 10.2 fold, the overall yield achieved 8.9%. The molecular of the purified laccase was understood to be 33.9KDa by SDS-PAGE. This also showed that the laccase was a single protein after purification. The optimal pH and temperature for the purified laccase were 4.5 and 55°C, respectively. The enzyme can keep high activility at 35-65℃. Moreover, the high laccase activity can be maintained at pH3-5. This enzyme have good temperature and pH stability and can basically meet the requirements of industrial production and utilization..Kinetic studies of laccase showes that the Km and Vmax is 0.032mmol/L and 8.7×10^-6 mol/Lï¹’ min when using the 2,2-azino-bis(3-ethylthiazoline-6-sulfonate) (ABTS) as substrate. But the Km and Vmax is 1.32mmol/Lå’Œ4.5×10^-6 mol/Lï¹’min when guaiacol as substrate. In the optimal reaction conditions, the effect of 2mmol/L of metal ions on enzyme activity was investigated. The result shows that Fe³⁺,Ag⁺,Mn²⁺ strongly inhibited the laccase activity. But it is not affected by Mg²⁺,Cl-,Na⁺,Ca²⁺. Some metal cation as Cu²⁺,Al³⁺,Zn²⁺ can significantly improve the activity of laccase, in which Cu²⁺ have best efficient..Chlorophenols and its derivatives which are typical toxic organic pollutants are discharged into environment mainly from effluent of chlorine- containing bleaching in the paper industry and biocides production process. In this work, 2,4-dichlorophenol (2,4-DCP) as typical chlorinated aromatic compounds was polymerized with catalyzer of laccase from a white-rot fungus, i.e. Trametes hirsuta. The polymerization process of 2, 4-diphenylphenol catalyzed by the crude laccase was analyzed. The results show that the processing time, reaction temperature and pH were important impact factors on the removal of 2,4-dichlorophenol. The optimum conditions are as follows: 35℃, pH 4.5. Under optimal conditions with aeration of 20hr, the removal efficiency of 2,4-dichlorophenol catalyzed by crude laccase can reach 93.6%. Furthermore, the crude enzyme was also separated and purified. The depurated laccase have the lower removal efficiency than the crude enzyme in the same reaction conditions.The molecular weight of the obtained products was analyzed with gel permeation chromatography (GPC). The result showes that the average molecular weight of the polymerization products is 2838KDa. The Chemical structure of the product was also analyzed by FT-IR and 13C-NMR. As a conclusion, the laccase from Trametes hrsuta has a high ability to polymerize 2,4-chlorophenols. It is suitable for wastewater treatment from chlorine-based bleaching process.The extracted DNA and RNA of Trametes hrsuta BYBF was detected by agarose gel electrophoresis to reach high purity. It meet the requirements of molecular biology of the gene fragments. Based on the conservation of laccase genes, the primers were designed, the cDNA of the laccase was cloned by RT-PCR, and connected with the target gene with the vector pMD 19-T and transformed into E.coli JM109. The positive clones were determined by blue and white spot experiment. The recovered fragment was used to determine sequence. It was gotten a length of 2154bp of nucleotide sequence. The sequence data was compared with the published sequence of relative fungi strains by ClastalW software, The results show that our strain is 99% identical to Pycnoporus sanguineus, Daedalea biennis, Polyporaceae and Pycnoporus cinnabarinus, and has little homology with Cerrena unicolor and Climacocystis borealis.