Isolating, Fermentation And Primary Application By Laccase From Tricholderma Harzianum ZF-2

Laccase is a type of multi copper-containing polyphenol oxidases capable of oxidizing a wide range of polyphenols and other lignin-derived aromatic compounds. Laccases can degrade lignin and phenoxy containing herbicide and waste water in petroleum industry and affect toxic phenolic materials; they can thus be widely used in paper pulp industry, food industry, medication and sanitation, biosensor and environment protection. So, the study on laccase possessed a certain the oretical and realistic significance.By the method of guaiacol plate screening, a strain of laccase producing microorganism was obtained from dote and soil samples collected from Shandong.(Termed as ZF-2).The strain was identified, and the medium components and fermentation conditions were optimized by response surface method, Brilliant Blue KN-R, Basic Violet and Congo red were decolorized by using crude enzyme, at the same time, the effects of the addition of redox mediators and the decolorization conditions on the decolorzation. The main results are as follows:1. Identification of strain ZF-2Based on its cultural characteristic, morphologic culture, 5.8S rRNA sequence analysis and phylogenetic tree analysis, ZF-2 was identified Trichoderma harzianum. The sequence of 5.8S rRNA obtained in this study has been registered at GenBank database and its accession number was HM051191.2. The optimization of medium and fermentation conditions of ZF-2With single factor was conducted, the study indicated that the best medium carbon source is wheat straw powder, the best nitrogen are (NH4)2SO4 and soybean power, metal ion is Cu²⁺ and inducer is Tween-20. Then determined the best C/N ratio by orthogonal test design. In the optimization of medium, the influences of four medium component on laccase production were frist evaluated using PB(Plackett-Burman)design. The path of steepest ascent was used to attain the optimal region of the medium composition. On the last step, the optimal concentrations of components were determined by central composite design and response surface analysis.The components of optimum medium for production of laccase were: wheat straw powder 7.6 g/L, soybean power 23 g/L, (NH4)2SO4 1 g/L, CuSO4 0.51 g/L, Tween-20 1 g/L, MgSO4 1 g/L, KH2PO4 0.6 g/L.The optimized fermentation conditions for production of laccase were: inoculum's volume, 2%; fermentation time, 42 h; fermentation temperature, 28℃; liquid volume, 50 mL/250 mL; intial pH, 6-8.The activity of laccase produced by ZF-2 which fermentated on the optimized medium and fermentation conditions got to 72.549 U/mL, 56.33 times higher than basal medium.3. The decolorization of different dyes by ZF-2 laccaseIn this part, preliminarily explore the roles of ZF-2 laccase on decolorization on azo dyes, anthraquinone dyes and triphenylmethane dye. The results showed that, when ZF-2 laccase deal with Congo red directly, decolorization rate got to 65.66% after 5 h; when ZF-2 laccase deal with Brilliant Blue KN-R and Basic Violet directly, decolorization rate got to 80.09 % and 26.09 % respectively after 4 h. Laccase collaborate with redox mediators (8-hydroxy benzopyridine, murexide, HBT ) decolorized three dyes, the results showed that, 8-hydroxy benzopyridine and murexide had no significant effects on the decolorization rate of three dyes, while added to HBT, the decolorization rates of three dyes were increased significantly: Brilliant Blue KN-R and Basic Violet react with laccase/HBT for 4 h, decolorization rate got to 92.87% and 71.32% respectively; Congo red react with laccase/HBT for 2 h, decolorization rate got to 79.03%.The results of decolorization conditions of three dyes as follows:The appropriate conditions of laccase degradating Brilliant Blue KN-R were: 50℃, pH 5.5, when laccase dosage was 15 U/mL, decolorization rate was higher than 85%.The appropriate conditions of laccase degradating Basic Violet were: 50℃, pH 5, when laccase dosage was 20 U/mL, reacting for 3 h, the maximum decolorization rate was 71.33%.The appropriate conditions of laccase degradating Congo red were: 50℃, pH 5, when laccase dosage was 20 U/mL, reacting for 2 h, the maximum decolorization rate was 85.34%.