| White rot fungi are a type of microorganism that can completely mineralize lignin in nature.This mainly depends on the unique lignin-degrading enzyme system secreted by white rot fungi,mainly including lignin peroxidase(LiP),manganese peroxidase(MnP)and laccase(Lac).Laccases can catalyze the cleavage of carbon-carbon and carbon-oxygen bond of lignin,play an important role in the degradation of lignin.It also has great application value and potential in environmental biotechnology such as the degradation of environmental organic pollutants.In this study,the white rot fungus Trametes sp.48424 was used to isolate and purify the laccase protein LAC-48-2 secreted by it.The enzymatic properties of LAC-48-2 and the effect of different metal ions and organic solvents on the activity and stability of LAC-48-2 were also investigated.Based on this,the ability of LAC-48-2 to degrade different structural chlorophenol compounds and to tolerate different metal ions and organic solvents during degradation were explored.Four laccase genes and their full-length cDNA sequences were cloned by degenerate primer PCR,thermal asymmetric interlaced PCR(TAIL-PCR),the cloned laccase genes were successfully expressed in the yeast Pichia pastoris.The main results of this paper are described as follows:When Trametes sp.48424 was cultured in GYP medium,laccase was induced by addition of 1mM copper sulfate.The highest laccase activity was observed on the ninth day of culture,and copper ion significantly increased the laccase production of Trametes sp.48424.The laccase protein LAC-48-2 was successfully isolated and purified by ammonium sulfate precipitation,anion exchange chromatography and hydrophobic chromatography.After purification,the specific activity of LAC-48-2 was 599.54 U/mg,and the molecular weight was about 52 kD.The enzymatic properties of purified LAC-48-2 were studied.LAC-48-2 had the highest affinity for ABTS,followed by 2,6-DMP and guaiacol.The optimal temperature and pH were 60℃ and pH 3.0.With the increase of temperature,the stability of laccase gradually decreased.LAC-48-2 had relatively strong tolerance to neutral or alkaline environments,and relatively weak stability under acidic conditions.The effects of different inhibitors,metal ions and organic solvents on the laccase activity and stability of LAC-48-2 were studied.Among the five inhibitors,DTT,NaNs,and mercaptoethanol had significant inhibitory effects on LAC-48-2.But the tolerability of LAC-48-2 to EDTA-2Na and SDS was relatively strong.Among the 14 metal ions,LAC-48-2 had strong resistance to metal ions such as Mn2+,Mg2+,Na2+,K+.High concentration of Cu2+ could promote laccase activity of LAC-48-2 to a certain extent.When 100 mM copper sulfate was added in the system,laccase activity was increased to 117.54%of the control.Fe2+ had the strongest inhibitory effect on laccase.Only 10 mM Fe2+ could completely inhibit the laccase activity.Among 11 organic solvents,LAC-48-2 was highly tolerant to glycerol and ethylene glycol,and relatively stable in glycerol,1,4-butanediol,propylene glycol.The purified laccase protein LAC-48-2 was used to degrade different structural chlorophenol compounds(3-chlorophenol,2,6-dichlorophenol,2,3,6-trichlorophenol)and 2-nitrophenol.LAC-48-2 had strong ability to degrade 2,6-dichlorophenol and 2,3,6-trichlorophenol.The 12-hour degradation rate of 800 mg/L 2,6-dichlorophenol by 1 U/ml LAC-48-2 could reach 99.17%,and to 200 mg/L 2,3,6-trichlorophenol was 96.91%.LAC-48-2 had a weak ability to degrade 3-chlorophenol,and LAC-48-2 could not degrade 2-nitrophenol.The degradability of LAC-48-2 for different substrates was as follows:2,6-dichlorophenol>2,3,6-trichlorophenol>3-chlorophenol>2-nitrophenol.LAC-48-2 still had a strong ability to degrade different chlorophenol compounds in the mixed chlorophenol(2,6-dichlorophenol+2,3,6-trichlorophenol).When 3-chlorophenol,2,6-dichlorophenol and 2,3,6-trichlorophenol were simultaneously present in the degradation system,the degradation rate of 3-chlorophenol by LAC-48-2 was still lowest,whereas the degradation rate of 2,6-dichlorophenol and 2,3,6-trichlorophenol still maintained a high level.The effects of different metal ions and organic solvents on the degradation of 2,6-dichlorophenol and 2,3,6-trichlorophenol by LAC-48-2 were studied.LAC-48-2 had relatively strong tolerance to metal ions such as Na+,Mg2+,K+,Mn2+,and Cu2+during the degradation of 2,6-dichlorophenol,but the tolerance to Fe2+ was poor.LAC-48-2 was highly resistant to metal ions such as Mg2+,Mn2+,K+,Na+,and Cu2+during the degradation of 2,3,6-trichlorophenol,80 mM and 100 mM manganese sulfate,potassium sulfate,sodium sulfate,copper sulfate had little effect on the degradation of 2,3,6-trichlorophenol.5%and 10%(v/v)of various organic solvents had little effect on the degradation of 2,6-dichlorophenol by LAC-48-2.LAC-48-2 had strong resistance to organic solvents such as isopropanol and 1,2-propanediol during the degradation of 2,6-dichlorophenol.5%and 10%(v/v)of various organic solvents also had little effect on the degradation of 2,3,6-trichlorophenol by LAC-48-2.LAC-48-2 was highly resistant to glycerol and ethylene glycol in the degradation of 2,3,6-trichlorophenol.This study also found that the mediator ABTS can greatly enhance the degradation of 2-nitrophenol and 3-chlorophenol by LAC-48-2,while another mediator AS(acetosyringone)had less effect on the degradation of 2-nitrophenol and 3-chlorophenol.Four different laccase full-length structural genes and their full-length cDNA sequences were successfully cloned by degenerate primer PCR,thermal asymmetric interlaced PCR(TAIL-PCR),including lac48-2(the full-length structural gene is 1996 bp,and the full-length cDNA is 1563 bp),lac48-9(the full-length structural gene is 2119 bp,and the full-length cDNA is 1563 bp),lac48-1(the full-length structural gene is 2110 bp,and the full-length cDNA is 1575 bp),lac48-3(the full-length structural gene is 2121 bp,and the full-length cDNA is 1560 bp).The lac48-2 gene encoded the purified laccase protein LAC-48-2 from Trametes sp.48424.Expression vectors for heterologous expression of four laccase genes in Pichia pastoris were constructed and introduced into Pichia pastoris GS115.The results of ABTS flat coloration,laccase activity assay and Native-PAGE of yeast fermentation broth all proved that four laccase genes could successfully express in the yeast and produce the active laccase protein in Pichia pastoris.The highest secreted laccase activity of four yeast transformants GS115(pPIC3.5K-lac48-2),GS115(pPIC3.5K-lac48-9),GS115(pPIC3.5K-lac48-1),GS115(pPIC3.5K-lac48-3)could reach 360.8 U/L,99.26 U/L,220.93 U/L and 65.08 U/L,respectively.The laccases expressed by GS115(pPIC3.5K-lac48-2)and GS115(pPIC3.5K-lac48-9)were able to efficiently degrade 2,6-dichlorophenol and 2,3,6-trichlorophenol.In summary,this paper focused on the lignin degradation enzyme laccase produced by the white-rot fungi Trametes sp.48424.Purification of laccase protein and its properties,degradation of chlorophenol contaminants by the purified laccase,cloning of laccase genes and their heterologous expression in Pichia pastoris were carried out in this study.The results showed that the purified laccase LAC-48-2 from Trametes sp.48424 had strong ability to degrade 2,6-dichlorophenol and 2,3,6-trichlorophenol,and had strong resistance to certain metal ions and organic solvents in the degradation of 2,6-dichlorophenol and 2,3,6-trichlorophenol.The results of this paper lay a good foundation for the better application of Trametes sp.48424 and its laccase in environmental biotechnology such as the efficient degradation of environmental pollutants. |
PDF Full Text Request