Isolation,purification,mediator System And Pollution Degradation Applications Of Laccase From Trametes Hirsuta

Laccase?EC 1.10.3.2?is a polyphenol oxidase containing multiple Cu²⁺,which is found extensively in fungi,bacteria,higher plants,and insects.Laccase can catalyze aromatics and other substances,such as lignocellulose,dyes and pesticides.Since the redox potential of some environmental compounds are higher than laccases,it restricts laccase application on degradation fields.The discovery of mediator widens the role of laccase and enhances the degradation rate.Laccase/mediator system?LMS?can degrade non-phenolic compounds and compounds with high redox potential.Therefore,laccases demonstrate great potential applications on degradation of lignin and environmental pollutants.In the present study,26 strains of wild fungi were used to screen objective stains with high resistance for pesticides.A strain?numbered as BUA-01?was obtained and further studied including culture and enzyme production,extracellular laccase isolation and purification,enzymatic properties and gene cloning,and LMS degradation towards pollutants.The details are as follows:1.Screening of objective strains were performed using the 26 isolated fungal strains and the pesticides targets of imidacloprid?0.5%?,acephate?0.2%?,glyphosate isopropylamine salt?0.4%?and cypermethrin?2.2%?.The BUA-01 strain was obtainsed with the growth rates of 6.75±0.25 mm/d,5.25±0.00 mm/d,3.25±0.13 mm/d,and 4.67±0.26 mm/d in the above four screening media.Based on both morphological observation and ITS molecular identification,the strain was identified as Trametes hirsuta.Culture condition study revealed that the optimal condition for the mycelia growth included starch as the carbon source,soybean powder and yeast extract as the nitrogen source,40/1 and10/1 as the C/N rate,temperature as 37°C,and pH as 6.0-7.0.The assayed growth factors had no significant effect on mycelial growth.It demonstrated high laccase activity in liquid fermentation.The highest extracellular laccase activity of 1081.33±6.3 U/mL was obtained in the broth with a Cu²⁺adjunction concentration of 0.25 mmol/L after a 96 h of cultivation.It was about 26-fold of that of the control group.Fermentation of the present strain also manifested significant decolorizing activities towards azo dyes evans blue,methyl orange,and eriochrome black T with the decolorization rates at 12h of 93.31±0.16%,92.37±0.42%,79.25±0.64%,respectively.2.Fermentation of the present strain was performed with 5%inoculum and a final concentration of0.25 mM Cu²⁺,and cultured at 28°C and 180 rpm in a shaker for 3 days.Crude enzyme solution was futher purified following three ion-exchange chromatography steps of DEAE-Sepharose,SP-Sepharose and Q-Sepharose.The purified laccase?THL?was determined to be 67.7 kDa by SDS-PAGE.When ABTS was used as the substrate,it manifested optimal pH and temperature of 2.2 and 60°C,respectively.It possessed considerable high thermo and pH stabilities at 50-65°C and 4.2,respectively.The K_m and V_max were determined to be 28.44?M and 555.56?mol/min,respectively.Mg²⁺,Ca²⁺,Mn²⁺,Cu²⁺,Cd²⁺,Co²⁺,and EDTA?10.0 mM?can significantly inhibit the enzyme activity with the inhibitory ratio of 25%-58%.Moreover,Na⁺at the concentration of 1.25 mM can softly enhance the enzyme activity of about 10%.K⁺at the concentration of 1.25-10 mM had no significant effect on the laccase.By using a degenerate primer to amplify the conserved sequence of laccase,a partial laccase cDNA sequence with a length of 1226 bp was obtained.The similarity to T.hirsuta?KU878364.1?was 97%and the genetic distance was 0.072.3.LMS studies revealed that the most suitable mediators of the present laccase were acetosyringone?AS?and tetramethylpiperidine nitrogen oxide?TEM?.LMS of THL had the best degradation effect on basic fuchsin,methyl orange,malachite green and Evans blue.The highest degradation rates were 72.3%,86.1%,88.2%and 89.2%at 24 h,respectively.The results of the full-wavelength scan showed that THL-AS has a greater ability to enhance the reaction than that of THL-TEM.Both of them generated new substances during the degradation of basic fuchsin,methyl orange and Evans blue dyes.The effect of pH,temperature,and mediator concentration on LMS showed that THL-AS was very stable at pH 2.0-5.0,20-60°C with the mediator concentration of 0.1 mM.It can effectively degrade the dyes over 80%in 1 h.THL-TEM was stable at pH 3.0-4.0,50-60°C and mediator concentration of 0.5-1.0 mM,while its degradation efficiency was not as high as that of THL-AS.At 24 h,THL-TEM possessed the highest degradation ratio on acephate of 65.56%,while THL/ABTS showed the best degradation effect on aflatoxin B₁ of 20.88%.The 10-fold and 50-fold dilutions of the LMS reaction solution had low phytotoxicity to ryegrass,and the effect on root length growth was smaller with increasing dilution.