Gene Cloning And Expression Of White Rot Fungi Versatile Peroxidases

The effective degradation of lignin by white-rot fungi mainly relies on a series of lignin degrading enzymes,in which the versatile peroxidase?VP?is a heme-containing peroxidase.VP combines the properties of both lignin peroxidase?LiP?a nd manganese peroxidase?MnP?and has great application value and pote ntial in the case of lignocellulose bio-pretreatment,dye decolorization,pollutant degradation and transformation.Therefore,the discovery of genetic resources,the construction of efficient expression system and the exploration of the regulation rules of VP can help to further reveal the lignin degradation mechanism by White-rot fungi and its application research.Based on this,the versatile peroxidases gene of Physisporinus sp.P18 were studied by cloning,heterologous and induced expression in this paper and the main conclusions are as follows:The molecular biological property of strain P18 in our laboratory was indentified and classified as Physisporinus sp.P18.The research of enzyme production rule showed that Mn²⁺could promote the extracellular enzyme activity of Physisporinus sp.P18 Mn²⁺dependent peroxidases and the higher concentration of Mn²⁺in the range of 0¹mM,the more obvious the promoting effect was.Furthermore,the full length structure and encoding genes of VP1 and VP2 were obtained by degenerate PCR and Tail-PCR and the bioinformatics analysis was performed of which the full length structure genes of VP1 and VP2 are 1669bp and 1651bp and both encoding gene without introns are 1077bp which encoding 358 amino acids?88%homology with each other?;the predicted versatile peroxidases coding sequences contained 21aa signal peptides,the molecular weight of mature proteins without signal peptides are 36.83KD and 35.79KD and isoelectric points are 4.54 and 4.35 respectively.The promoting effect of Mn²⁺on the transcription level of Physisporinus sp.P18versatile peroxidases were studied by real-time fluorescence quantification PCR and the effect rule of Mn²⁺on the expression of versatile peroxidase was explored preliminarily.Mn²⁺had a significant effect on the transcription level of Physisporinus sp.P18 VP1³ in the low concentration range of 150⁵00?M,but the optimal concentration of different VP genes was different that 150,300,500?M Mn²⁺has the best promotion effect to VP1³respectively with 4.0,3.5,3.8 times promotion;when Mn²⁺increased to 1000,1500?M high concentration,it still had 2.3 and 3.7 times significantly promotion effect to VP3respectively.Physisporinus sp.P18-rVP1 expression vector was constructed and expressed in Escherichia coli.The active recombinant protein was obtained by refolding and renaturation of inclusion body and then purified by nickel affinity column.Finally,the yied of purified rVP1 was 18mg/L and the specific enzyme activity was 23.1U/mg.The characterization and function study results of purifired rVP1 showed that,when the substrate was Mn²⁺,the optimum reaction pH was 5.0,the optimum reaction temperature was 40°C;Mn²⁺could affect the kinetic constants of rVP1 to other substrates;and rVP1had good tolerance to neutral and alkaline environment,most metal ions and enzyme inhibitor SDS.In the presence of pH 5.0 and Mn²⁺,rVP1 had strong decolorization effects on five different types of dyes and can catalyze industrial alkaline lignin and natural lignin EMAL effectively.In this paper,the genes of Physisporinus sp.P18 versatile peroxidases were obtained,the induction effects of Mn²⁺to VP transcriptional expression were explored preliminarily,the expression system of E.coli was constructed and the active recombinant rVP1 was obtained and characterized.This paper lays the foundation for further study on the structure and function of Physisporinus sp.P18 versatile peroxidases,and also provides a basis for revealing its molecular mechanism of expression regulation and lignin degradation.