| With the rapid development of the printing and dyeing industry,the large-scale use of synthetic dyes has caused great damage to the environment,and it is urgent to explore a degradation method with obvious decolorization and low cost.White rot fungi can secrete abundant non-specific extracellular enzymes,which can degrade toxic and harmful synthetic dyes into low-toxic or non-toxic chemical substances,and alleviate environmental pollution caused by dyes.The extracellular oxidase enzymes secreted by white rot fungi mainly includes 3 kinds of enzymes,Lignin peroxidase(Li P),Manganese peroxidase(Mn P)and Laccase(Lac).Among them,laccase plays an important role in the degradation of dyes.Therefore,this study screened high-yielding laccase strains,optimized their enzyme production conditions,and applied the obtained high-yield laccase strains to the decolorization experiment of synthetic dyes.It expects to be used as engineering candidate strains for laccase production to solve the environmental problems caused by synthetic dyes.The main results obtained in this study:1.The best laccase-producing strain,Auricularia cornea AC5(SDAU-AC-005),was determined based on the characteristics of the brown-red oxidation circle produced by 22 strains on the guaiacol selection medium and the extracellular enzyme activity of the liquid strain.2.Studies on the best culture conditions for A.cornea AC5.The most suitable carbon source for solid medium was maltose,the most suitable nitrogen source was yeast powder,and the most suitable pH for culture was pH 6.0.Comprehensive analysis of growth rate and growth conditions,the suitable temperature was 26℃.The enzyme activity was highest in the fermentation condition when rotation speed of 160 r/min,inoculation amount of 5 pieces of0.7 cm mycelia,temperature of 26 ℃.3.A.cornea AC5 mainly secretes laccase and lignin peroxidase.The laccase activity of A.cornea AC5 was significantly higher than that of manganese peroxidase and lignin peroxidase.The results showed that wheat bran extract can effectively improve the laccase activity of A.cornea AC5.The optimal production conditions of laccase were obtained by response surface method.The laccase yield can reach the highest,787.87 U/m L,when culture time 6 d 22 h,culture temperature 26.9℃,10% wheat bran extract,at pH 5.Within the range of the level of each factor,the response sequence of the main factor was: A(cultivation time)>D(pH)>C(wheat bran extract concentration)>B(temperature).4.The decolorization results of different dyes by A.cornea AC5 showed that this strain had a certain broad spectrum for the decolorization of dyes,and it had the best decolorization effect for the triphenylmethane dye malachite green.The decolorization rate of 75 mg/L malachite green for 12 h can reached 83.27%.5.The response surface method was used to study the influence of different factors on the decolorization of dyes.The single factor experiment was carried out through different dye addition time,temperature,dye concentration and pH.The response surface method was designed by selecting the central point.The results showed that the decolorization rate of51.45 mg/L malachite green was 88.57% when addition time 7 d 10 h,pH 5.7,temperature26.41 ℃.Within the range of the level of each factor,the response sequence of the main factor was: B(temperature)>A(incubation time)>C(dye concentration)>D(pH).6.A laccase gene Cluster-6377.0 of A.cornea was obtained by transcriptomic data.The docking prediction with different dye molecules shows that different dye molecules can be effectively combined to degrade the dye.7.The toxicity test results of malachite green dye on garlic show that the dye inhibits the growth of garlic to a certain extent,caused the chromosome aberration of garlic root tip cells during mitosis,and reduced the mitotic index at the same time.The growth of garlic cultured with degraded malachite green was not significantly affected,indicating that the toxicity of the degraded malachite green dye was significantly reduced. |
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