Screening Of Laccase Producing Strain And Study On Its Plasma Mutagenesis

Laccase is a copper-containing polyphenol oxidase that can oxidize a variety of compounds,especially phenolic compounds,while reducing molecular oxygen to water.Due to its high redox potential,laccase has great application potential in many aspects such as chemical synthesis and environmental treatment.At present,the main producing strains of laccase are filamentous fungi,among which white rot fungi are in the majority.However,the current use of filamentous fungi to produce laccase still has shortcomings,that is,filamentous fungi have a longer fermentation cycle and lower enzymatic activity of laccase,which leads to higher production costs of laccase.In response to this problem,this thesis has carried out the following research work:(1)A fungus Trametes sanguinea with high laccase activity was screened from white rot wood samples from different areas.Then,the strain was identified by 18S and ITS-5.8S r DNA sequences and named as Trametes sanguinea WTFA5.The laccase activity of this strain was reached 123.13±10 U/L after 10 days of fermentation in the re-screening medium.Using its genome as a template,PCR was performed with degenerate primers for the copper ion binding domain of laccase.Three different laccase encoding genes were amplified and named Lac 1,Lac 2,and Lac 3,respectively.Among these three enzymes,Lac 1 was purified and its specific enzyme activity was 297.76 U/mg,K_mand K_catwere 58.76±5.33?M and 13.89±0.01 s^-1,respectively.Subsequently,the spores of Trametes sanguinea WTFA5 were used for plasma mutagenesis,and mutants such as S3 and S4 were obtained.Among them,the laccase activity of S4 strain reached 228.5 U/L when fermented in the re-screening medium for 9 days.Compared with the wild-type strain A5,the production time of S4strain was 1 day earlier,and the activity of laccase was increased by 84.3%.(2)The culture conditions of S4 strain were optimized.First,the carbon and nitrogen sources were screened through single factor experiments.On this basis,the appropriate concentration range of carbon,nitrogen,inducer,and copper ion were selected.The orthogonal experiment design was carried out based on the above-mentioned concentration interval of the highest laccase production,and the optimization experiment was carried out according to the orthogonal experiment design.The optimization experiments was performed according to the orthogonal experiment design.The optimal medium composition is glucose 40 g/L,soybean meal powder 20 g/L,2,5-dimethylaniline 20.42?M,copper sulfate pentahydrate 6 m M and other elements.The laccase activity of S4 strain was reached 40420±851.6 U/L under this medium.(3)Genome resequencing analysis of wild strain A5 and mutant S3 and S4 strains.First,the whole genome of strain A5 was sequenced and the chromosomal framework was constructed.Then,the genomes of strains S3 and S4 were compared with strain A5 by genome resequencing technology.After preliminary analysis,the results showed that the sequence within 4 kb upstream and downstream of the Lac1 gene in the mutant did not change.The change in laccase activity in the mutant may be caused by other mutations.