Decolorization Of Synthetic Dyes And Heterologous Expression Of Manganese Peroxidase From White Rot Fungus SQ01

White rot fungus is a kind of fungus specially used to degrade lignin,cellulose,semi-fiber and other substances.The previous study found that extracellular enzymes secreted by white rot fungus SQ01 can transform phenols,non-phenols and biphenyl intermediate metabolites.It also has obvious decolorization effect on triphenylmethane dyes.Due to both long growth cycle and enzyme production cycle of white rot fungi,expanding the scope of dye decolorization,optimizing decolorization conditions and improving its expression level have become the focus of current research.This is helpful to further develop the potentiality of its industrial application.In this study,we studied the decolorization ability of manganese peroxidase(Mn P)secreted by white rot fungi SQ01 to azo dyes by single factor method.The conditions of biological decolorization were optimized by response surface method.On this basis,the bleaching and decolorization of cotton fabric dyed with thiazine dyes was studied.The results showed that the factors affecting the biological decolorization of Mn P were in the following order: enzyme concentration > dye concentration > temperature >p H value.The optimal biological decolorization conditions after response surface optimization were as follows: the temperature was 49°C,the p H value was 4.6,the dye concentration was 85.5 mg/L,enzyme concentration was 430 U/L,and the decolorization rate was 84.5%.The decolorization rate of cotton fabric dyed with thiazine dyed by Mn P for 2 h was 16%.The results showed that the Mn P has potential application value in the field of biological decolorization of azo and thiazine dye wastewater.In order to use bioinformatics to analyze and predict the gene sequences of manganese peroxidase isozymes secreted by white rot fungi SQ01.We extracted its transcriptome.Then sent it to Shanghai Sangon Biotech company for sequencing.Seven full-length manganese peroxidase isozyme sequences were screened by comparing the sequences in NCBI BLAST after sequencing.And then carry on the bioinformatics analysis to the isozymesequences.The results showed that the contents of G and C in gene sequences of Mn P isozymes were higher than 60% and between 60% and69%.They were difficult to be denatured by high temperature and alkalinity.The molecular weight range of isozymes were between 37.9k D-46.0k D.The contents of neutral amino acids were more than 59%.These isoelectric point were between 4.18 and 6.13,which is slightly acidic.The phylogenetic analysis of Mn P isozymes by MEGA5 software showed that the gene sequences of 7546_t,7547_t and 8963_t were 100% similar to the known sequences XM008043881.1,XM008044277.1 and XM008039971.1,respectively.The signal peptides of isozymes were analyzed by Signal P4.0software.We found that all Mn P isozymes have signal peptides at the N-terminal,which belong to secretory proteins.Subcellular localization of isozymes was carried out by Target P Server.It was predicted that sequences of 7546_t,7547_t,7548_t and 14564_t were more likely to have signal peptides of secretion pathway,which were consistent with the results of signal peptide analysis.In addition,Mn P isozymes had heme binding sites,substrate binding sites,manganese and calcium binding sites.In order to improve the expression of Mn P,primers were designed with c DNA as template for PCR amplification.The target sequence was ligated with plasmid p MAL-p2 x to form a recombinant plasmid p MAL-p2x-mnp,which was successfully expressed in E.coli BL21.The recombinant protein Mn P-MBP was purified by Amylose column and digested with Prescission Protease.Then a new band appeared,which was speculated to be the target protein Mn P.The enzyme activity was 0.5 U/m L.Enzyme activity was relatively low.In order to increase the enzyme activity of Mn P,we intended to express the target gene in Trichoderma reesei Tu6.We have constructed the recombinant plasmid p SKPss M successfully.We also prepared the protoplasts of Trichoderma reesei strain Tu6.The basic preparation work was made for transforming the gene sequence with strong promoter ? signalpeptide and terminator sequence into the protoplast of Trichoderma reesei Tu6 for heterologous expression.To sum up,the biological decolorization of azo and thiazide by manganese peroxidase secreted by the white rot fungus SQ01 was studied.Due to the low content of Mn P in wild bacteria,its application in industry is limited.We analyzed the gene sequences of manganese peroxidase isozymes by bioinformatics and studied the heterologous expression of manganese peroxidase.Mn P was successfully heterologous expressed in E.coli.Because the activity of recombinant enzyme is relatively low,we planed expressing Mn P in Trichoderma reesei Tu6.The study laid a foundation for further expanding the application of Mn P in the field of biological decolorization.The study also laid a research foundation for increasing the output of Mn P and applying recombinant enzyme in the field of industrial decolorization.