Isolation And Purification Of Laccase From The White Rot Fungus Leiotrametes Lactinea And The Enzymatic Activity In Dye Degradation

In the process of production and use of dyes will produce a large number of waste water,these waste water contains a large number of complex structure,difficult to degrade and biotoxic compounds,to the water and soil ecological environment has brought serious pollution.At present,the physical adsorption separation and chemical oxidation in dye wastewater treatment technology have the disadvantages of high cost,difficult operation and easy to produce secondary pollution.Laccase,as a kind of copper containing polyphenol oxidase,has many advantages in dye degradation,such as easy operation,less secondary pollution and a wide range of substrates.In this study,Leiotrametes lactinea strain(JZ-21)was used as the experimental material,and its mother culture medium and liquid fermentation medium for laccase production were optimized.Then,laccase was isolated and purified by ion exchange chromatography.The physicochemical properties of laccase and its degradation effect on dye were further studied.The following conclusions are drawn in the end:1.After optimization,the optimal culture conditions of JZ-21 mother culture medium were as follows:xylose 20 g/L,yeast extract 4.57 g/L,optimal C/N ratio of 25:1,rice bran 5g/L,Mg SO₄·7H₂O 1g/L and KH₂PO₄ 1g/L,the optimal culture temperature was 40?;The optimum conditions for laccase production by liquid fermentation were as follows:potato 200g/L,glucose 20g/L,KH₂PO₄ 3 g/L,Mg SO₄·7H₂O 1.5 g/L,sawdust 20 g/L,rotation speed 160r/min,26?,p H 6,inoculation quantity of 7 pieces of mycelium.2.After separation and purification of laccase by DEAE-Sepharose,Q-Sepharose and DEAE-Sephadex ion exchange chromatography,the electrophoresis pure laccase with a molecular weight of 68.5 k Da was obtained.The final purification ratio was 15.5 times,and the recovery rate of laccase was 18.3%.3.The enzymatic properties of the obtained pure laccase were analyzed.The optimal temperature range of the enzyme was 40?to 60?,and the tolerance of the enzyme was best at 30?,and the activity of laccase could be kept stable at 20?to 50?.The optimum p H is 2,and the tolerance is best at p H 5 to 6.Hg²⁺and Al³⁺had significant inhibitory effects on laccase activity at the three concentrations,Na⁺,Zn²⁺,Mn²⁺and Ca²⁺had slight inhibitory effects on laccase activity,and the inhibitory effect gradually increased with the increase of concentration.Cu²⁺had similar inhibitory effect on laccase activity at the three concentrations,while K⁺had no obvious effect on laccase activity.1%methanol and ethanol had no effect on the activity of laccase,while 1%acetone and isopropanol had slight inhibition on the activity of laccase.When the concentration of the above four organic solvents was 10%,the activity of laccase could still be maintained above 75%.4.In the experiment of dye degradation by laccase,it was found that laccase could degrade many kinds of dyes,and the degradation rate of reactive black and coomassie bright blue was obviously improved after the addition of ABTS and syringaldehyde.The degradation rate of reactive blue R,Evans blue,chrome black T,triphenyl blue,crystal violet,malachite green,coomassie brilliant blue,basic magenta,bromophenol blue,bromothymol blue and reactive brilliant blue was more than 80%,and the degradation rate of methyl orange was more than 60%.After optimized by response surface methodology,degradation rates of reactive red KD-8B and reactive navy blue were increased by 38%and 15%,respectively.5.The degradation products of malachite green dye by JZ-21 laccase were further analyzed.Malachite green under the action of laccase into leucomalachite green,after the decomposition of leucomalachite green into m-coumaric acid,4-dimethylaminobenzoic acid ethyl ester and p-isopropyl aniline,4-dimethylaminobenzoic acid ethyl ester can also be further decompose into N,N-dimethylaniline.In conclusion,this study provided a basis for the study of laccase properties and dye degradation of Leiotrametes lactinea,which has a certain research value.