Mutation Breeding Of Trametes Sp. From Bamboo Forest And Optimization Of Enzyme Production Conditions

Laccase is the main degrading enzyme which is an extracellular enzyme in the lignin-degrading enzyme system.It can catalyze the benzene ring to opening and degrade acidic substances.The state of the soil acidification of bamboo forest land can be alleviated by this enzyme to a certain extent.Through the preliminary screening work,the high laccase-producing Trametes sp.27-1 have been screened in the bamboo forests.The laccase activity of the strain is much higher than that of the similar strains reported before,which reaches 75.45U.This kind of strain has good degradability to bamboo lignin.Thus,by means of mutagenesis and breeding of the strain,a stable and high-yield strain is obtained.Finally,the laccase production was increased,and the degradation ability of the strain to lignin was improved too.?1?Ultraviolet irradiation combined with nitroguanidine was used for mutation breeding of Trametes sp.27-1.The mutant strains with stable and high yield were screened by observing the performance of the mutant strains on the culture plate and re-screening the laccase activity of the mutant strains after the initial screening.During the initial screening on the guaiacol plate,10 strains were screened out according to the degree of discoloration and the diameter of discoloration circle.After rescreening the enzyme activity,positive mutation was found in 8 strains out of 10,and genetic stability of 3 of them was studied.Finally,the mutant strain ZB-563 was obtained.The laccase activity of ZB-563 reached 95.15 U,which was 21.26%higher than that of the original strain.It was a stable strain with high production of laccase.?2?The optimum culture conditions of strains were explored by changing the single factors such as culture time,temperature,rotating speed,inoculation and liquid loading.The results showed that 12.5%seed solution was inoculated into 250 mL triangular bottle with 150 mL liquid content,and 200 r·min^-1shaking culture could achieve the optimum condition at 30?.?3?By response surface analysis,the optimum liquid fermentation broth composition of the strain was determined as glucose 13.69 g·L^-1,copper sulfate 0.5 g·L^-1,pH 4.33,peptone 10 g·L^-1,manganese sulfate 0.4 g·L^-1,potassium dihydrogen phosphate 2 g·L^-1,magnesium sulfate 0.5 g·L^-1,calcium nitrate1 g·L^-1,Tween80 2 g·L^-1.Under the optimum conditions,the enzyme activity of the mutant strain reached129.68 U in the incubator,which was 71.87%higher than that of the original strain.?4?The study on the actual degradation ability of ZB-563 showed that the mutant strain ZB-563could significantly improve the weight loss and lignin degradation rate of the matrix compared with the original strain.It further showed that the mutant strain had better degradability than the original strain and could degrade bamboo lignin better.In the degradation of bamboo leaves,the accumulative degradation rate of strain ZB-563 was 30.93%in 40 days,which was higher than that of Phanerochaete chrysosporum.A large number of strains were re-planted in the original bamboo forest to help remediate the microbial community in the soil.Because of its strong enzyme-producing ability after mutation and breeding,it can improve the degradation efficiency of the organic matter in the forest land and slow down the degradation of the forest land.The strain can also transform tender bamboo and bamboo shoot shell into green storage feed,degrade lignin to improve feed palatability and feed quality.It can also degrade a large amount of lignin in the cooking wastewater produced by bamboo processing,and improve the efficiency of sewage treatment.