The Study Of Culture Conditions' Optimization And The Laccase Of Lepista Sordida

In recent years,a large number of studies have proved that laccase has the activity of degrading dyes and pesticides,and it has a broad application prospect in sewage treatment and environmental remediation.In this study,the strain JZ05 was used as the experimental material to select and optimize the parent culture medium formula and culture conditions.The liquid fermentation conditions of laccase production were optimized.Then,using the fermentation broth as the crude enzyme,the purification methods such as ion exchange chromatography were used to isolate,purify and identify the laccase from tricholoma hualian.At last,the degradation effect of fermentation broth and laccase on the dye was studied.Through the above experiments,the following results are obtained:1.The optimal carbon source of mother species of Lepista sordida was cornmeal,the nitrogen source was peptone,the optimal carbon to nitrogen ratio of medium was 40:1,the optimal growth temperature of mycelia was 23?,and the optimal pH value of mycelia was7.0.The optimal culture conditions for liquid strains were:rotation speed of 150 rpm,inoculation quantity of 10 0.5 mm bacterial tablets,and culture temperature of 27?.The yield of laccase could be increased by adding cornmeal to liquid medium,and the optimal amount of cornmeal was 1.0 g/L.2.Liquid fermentation liquor of Lepista sordida as crude enzyme fluid,by optimizing the DEAE-Sephadex anion exchange chromatography,Q-Sepharose anion exchange chromatography,SP-Sepharose cation exchange chromatography and other methods,get electrophoresis pure level laccase of Lepista sordida,SDS-PAGE gel electrophoresis detection displayed as a single strip,according to the standard curve of the laccase protein molecular weight is 58.5 kDa.Combined with the ultrafiltration results of ultrafiltration tubes with different interception molecular weight,the laccase was determined to be a single subunit protein with a molecular weight of 58.5 kDa,which was named Lac-LS.3.Through the Lac-LS sequencing peptides sequence,get 488 sequences of peptides,including three peptides sequence?SLELVLGPGLGASGPHPFHLHGHTLSPYR QAGTFWYHSHGLSTQYCDGLR GPVGVTQCPLSPSHSFLYK?sequence of laccase conservative area,further illustrate the purpose of purification of proteins is laccase.Through the determination of the N-terminal sequence of Lac-LS,the amino acid sequence of its N-terminal part was obtained to be:A I G P I A D L H I,which had high homology with the N-terminal sequence of other fungal laccase.The 488 internal peptide sequences and N-terminal sequences obtained above were compared by BLASTP with the protein database predicted by the genome sequence of chlamydus citrini respectively,and the gene encoding Lac-LS was predicted as gene4851 through comparative analysis.According to the analysis,the laccase encoded by this gene contains 13 introns and 13 exons,which is similar to the number of introns in other fungal laccase.The molecular weight of the encoding laccase is 57kDa,the isoelectric point pI is 6.0,and the GRAVY value is-0.076,suggesting that the laccase is a hydrophilic protein.Through the analysis of conserved domain,the laccase encoded by this gene has three domains of typical fungal laccase:cu-oxidase₃,cu-oxidase and cu-oxidase₂.4.By determining the physical and chemical properties of Lac-LS,it is concluded that the optimum temperature of laccase is 60?;The optimal pH value is 3.0;It had good temperature tolerance in the range of 50?-60?,and the residual relative enzyme activity was more than 60%after 60?treatment for 60 min.Lac-LS was well tolerated in the pH3.0-ph 5.0.Metal ions had a certain effect on laccase of tricholoma auriculata:Na⁺,K⁺,Mg²⁺had no obvious effect on laccase activity,while Zn²⁺and Al³⁺had an inhibitory effect on laccase activity,and the inhibitory effect was gradually enhanced with the increase of concentration.Iron ion has obvious inhibitory effect below 1.25 mM,and has obvious promoting effect at 5 mM.5.Under the condition of same laccase activity,compare Lac-LS and fermented liquid of dyes?methyl red,methyl orange,chrome black T and coomassie brilliant blue?,the degradation effect of found Lac-LS for the degradation of dye rate reached thick enzyme fluid for dye degradation rate of more than 50%,in thick enzyme fluid on the degradation of dye in the degradation of laccase has played a major role.Then,Lac-LS with high laccase activity?2000 U/mL?was used for the degradation of dyes.The results showed that Lac-LS had degradation rates of over 90%for methyl red,methyl orange,chromium black T and coomassie blue.Methyl red and methyl orange dyes were then degraded by Lac-LS?3000U/mL?with higher enzyme activity,and then detected by HPLC.Both dyes were found to be completely degraded.The above research results will provide important data basis for the related research on laccase,especially for the degradation of dyes,and have certain research value.