Screening Mutants Of Coriolus Versicolor For Overproducing Laccase Under Nitrogen Nutrient Sufficiency And Enzyme-production Characteristics Research

The application of white-rot fungus relies on the lignin degeneration enzyme which has the widespread universality and the non-specific characteristic.However,the lignin degeneration enzyme is the secondary metabolite,and the synthesis of enzyme is stimulated by the lack of the nitrogen and other main nutrients.The activity of the Lignin degeneration enzyme appears only after the hypha stops growing.At present,the technique of the limited-nitrogen culture inevitably causes that the mycelium biomass and the enzyme activity is very low.For earlier application of White Rot Fungi and its lignin-degrading enzymes in the industry and environmental governance,this paper takes the fungus of Coriolus versicolor as the material,and hopes to obtain the mutant that produces the resistance of nutritional repression and synthesizes efficiently the laccase under the rich nutritional condition.By the ultraviolet mutagenesis,we breed the mutant of Coriolus versicolor which can produce the resistance of nutritional repression,and study its characteristics for producing the laccase.Its optimal media and culture conditions for the synthesis of laccase are also determined.And the decolorization and degradation effects of four refractory dyes of Pararosaniline,Congo red,malachite green and naphthol green B by the mutant strain in the nitrogen-limit and the nitrogen-rich liquid medium is further studied.The results are as follows:(1)We get a mutant strain CFCC-4 of Coriolus versicolor which can synthesize efficiently laccase in the nitrogen-rich condition.This mutant strain breaks the nitrogen restriction of the original strain for producing laccase.The production of the enzyme and cell growth can be achieved simultaneously in the nitrogen-rich condition.And laccase is the primary metabolite.The laccase activity of the mutant strain reaches 2822U/L in the static culture condition,increases 12 times compared to the original strain(211U/L).(2)The optimal medium and culture conditions of the mutant strain are identified as follows:Sucrose 20g/L,ammonium tartrate 2.2g/L,CuSO4 0.5mmol/L,surfactant Tween-80 1g/L,pH=4.5,adding guaiacol for a final concentration of O.Olmmol/L after cultured two days,shaking 12 days in the flask at 28?.At this point,laccase activity reaches 8572U/L,which is 3 times better after the optimization.(3)The decolorization and degradation effects of four refractory dyes of Pararosaniline,Congo red,malachite green and naphthol green B by the mutant strain in the nitrogen-limit and the nitrogen-rich liquid medium is further studied.At the same time,we explore the changes of dye absorption peaks.Pararosaniline,Congo red,malachite green and naphthol green B decolorized by the mutant strain have a much shorter decolorization time and higher decolorization rate than them decolorized by the original strain after culturing 6 days whether in N-riched or N-limited conditions.After decolorizing 36h,the decolorization rates of the four kinds of dyes by the mutant strain are very high,reached more than 92%.The decolorization rate of Naphthol Green B(100%)is highest,followed by malachite green(97.2%)and Congo red(95.8%),and the decolorization rate of Pararosaniline(92.5%)is lowest.The height order of the decolorization rates by the mutant strain is:naphthol green B>malachite green>Congo red>Pararosaniline.However,for the original strain the decolorization rate of Congo red is highest only for 93.4%,but the decolorization rates of other three dyes is less than 40%.The degradation rates of four different dyes by the mutant strain are significantly higher than that by the original strain in nitrogen-rich and N-limited conditions.After degrading 36h,for the mutant strain all of the degradation rates reach more than 88%.The degradation rate of Naphthol green B is highest for 99.5%.The height order of the degradation rates by the mutant strain is:naphthol green B>malachite green>Congo red>Pararosaniline.However,for the original strain the degradation rate of Congo red is highest only for 75.8%,but the decolorization rates of other three dyes is less than 40%.After treatment by the mutant strain,the characteristic absorption peaks of the dyes Wastewater medium move significantly to the short-wave compared to untreated dye solution,and the absorbance of the characteristic absorption peak decreased significantly.This shows that the structure of dyes has changed,which is degraded by enzymes.