| Lignocellulose is a recyclable biology resource with large reserves in nature.Degradating lignocellulose into monosome that can be used by biology and converting monosome in to fuel ethanol draws more and more attention.Lignocellulose is a highly crystalized macro molecule consists of cellulose,hemicellulose and lignin.It's very difficult to be degradated,so some physical,chemical and biological methods must be used for degradation.White-rot fungi is the only biology confirmed that can completely degrade lignin.In this experiment,6 strains of white-rot fungi were used to compare the relative enzyme systems on lignocellulose degradation.2 of the strains with relatively higher ligninase activity were selected as the initial strains for breeding.By usingmutation and protoplast fusion,a fusion strain with entire ligninase system and higher enzyme activity was obtained.It can be used as reference and experiment strain for further research.1.By comparing the relative enzyme systems on cellulose and lignin degradation of these 6 white-rot fungi strains,we found that the liginase activity of CICC40719 and CICC14076 were relatively higher.Strain ACCC30942 was capable of secreting 3 enzyme components of cellulose,the rest strains could secrete part of the cellulose.The ligninase of white-rot fungi was stable at 40-50'C with pH3.5-5.0 ands the enzyme activity was higher,while the cellulose was stable at 50-60? with pH5.0-6.0 and the enzyme activity was higher.By comparing the enzyme activity,CICC40719 and CICC 14076 were selected as the initial strains for mutation and dreeding.2.UV was introduced to process the spore suspension of CICC40719 and CICC 14076,.HC value was regarded as the standard for primary screen,enzyme activity of flask fermentation was regarded as the standard for secondary screen.Mutant strain UA-4-08 which has higher Lac activity,and mutant strain UB-5-02 which has higher MnP and LiP activity were obtained.The Lac activity of UA-4-08 was 6.22U/mL,which was 49.88%higher than its initial strain CICC40719.Variance analysis shown a significant difference.The MnP and LiP activities of UB-5-02 were 53.40U/mLand 48.82U/mL respectively,which were 32.47%and 31.10%higher than its initial strain CICC 14076.Variance analysis shown a significant difference.3.NTG was used to process the spore suspension of CICC40719 CICC14076,HC value was regarded as the standard for primary screen,enzyme activity of flask fermentation was regarded as the standard for secondary screen.Mutant strain NA-4-05 which has higher Lac activity,and mutant strain NB-5-05 which has higher MnP and LiP activity were obtained.The Lac activity of NA-4-05 was 6.12U/mL,which was 47.76%higher than its initial strain CICC40719.Variance analysis shown a significant difference.The MnP and LiP activities of NB-5-05 were 49.21U/mL and 43.54U/mL respectively,which were 22.07%and 16.91%.Variance analysis shown a significant difference.4.UA-4-08?UB-5-02 and NA-4-05?NB-5-05 were chosen as the parent strains and were processed by helicase to get protoplast.The protoplasts were processed by heat and UV.PEG was used to promote the fusion.Fusion strain F-5-07 with higher ligninase productivity was finally obtained.The Lac,MnP and LiP activities of this fusion were 8.79U/mL,57.51U/mL and 58.58U/mL respectively,compared with the initial strains,there was a significant difference.After a 5-generation continuous passage,the ligninase activity of each generation was examined.The results shown that after culturing for 5 generations,the Lac,MnP and LiP activities didn't reduce.The ligninase components,optimized temperature,optimized pH,thermal stability and pH stability shown no difference compared with the initial strains CICC40719 and CICC14076.Conclusions of the paper are as follow:(1)Among 6 white-rot strains,CICC49719 has the highest Lac activity,CICC14076 has the highest MnP and LiP activities.The optimized reaction temperature of relative enzymes for lignocellulose degradation of these 6 strains is 40-50?,the optimized pH is 4.0-4.5,in the environment with temperature which is lower than 60? and with pH which is lower than 5.5,the enzymes show a good stability.(2)UV was introduced to process CICC40719 and CICC 14076,mutant strains UA-4-08 and UB-5-02,which have relatively higher ligninase activities are obtained.The Lac activity of UA-4-08 is 49.87%higher than its initial strain.The MnP and LiP activity of UB-5-02 are 32.47%and 31.10%higher than its initial strain.(3)NTG was used to process CICC40719 and CICC14076,NA-4-05,which has a higher Lac activity and NB-5-05,which has higher MnP,LiP activities were obtained.The Lac activity of NA-4-05 is 47.47%higher than its initial strain.The MnP and LiP activities of NB-5-05 are 22.08%and 16.91%higher than its initial strain.(4)Protoplasts of UA-4-08 and UB-5-02 are inactivated by heat,protoplasts of NA-4-05 and NB-4-05 are inactivated by UV,PEG is used for promoting the fusion process.F-5-07,which has a higher liginanse activity is obtained.A 5-generation continuous passage shows that fusion F-5-07 is genetically stable.The enzyme characteristics of F-5-07 show no difference compared with initial strains CICC40719 and CICC14076.The innovation points of the passage are:(1)For the first time,the enzyme components and characteristics of relative lignocellulose degradation enzyme of multiple white-rot fungi strains was completed.It could provide reference with the further development and application of strains.(2)The experiment indicated that mutation is helpful in obtaining mutant strains with higher enzyme activities and UV is better than NTG(3)UA-4-08,UB-5-02,NA-4-05 and NB-5-05 are selected to prepare for inactivated protoplasts for multi-parent fusion,fusion F-5-07 with higher Lac,MnP and LiP activities is obtained.Within 5 generations,this fusion strain is genetically stable and can be used as the experiment strain for further development and application. |
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