Heterologous Expression And Characterization Of VPL1 Gene In Lentinula Edodes

The biodegradation of lignocellulosic biomass has been widely studied.It is reported that among the organisms that can modify lignin in nature,basidiomycetes white rot may have developed the most effective oxidation system so far.It is a system based on the function of extracellular oxidoreductase to complete lignin decomposition.It has this system in Lentinula edodes and Pleurotus eryngii,Through the in-depth study of the whole process mechanism of lignin degradation catalysis,it is found that the multifunctional peroxidase versatile peroxidase(VP)in lignin degradation enzyme system has gradually become a research hotspot in lignin degradation enzyme system recently because of its wide substrate catalytic characteristics and low catalytic conditions,showing great application value and prospect.And VP has the characteristics of both lignin peroxidase(Li P)and manganese peroxidase(Mn P).The in-depth study of VP will help to reveal the catalytic mechanism of lignin degradation.Therefore,this paper studied the enzyme production law of Pleurotus eryngii 3403 and Lentinula edodes strains Gsm207,Bsm83,A2sm99 and 215sm280 in the laboratory.It was found that nitrogen source limiting culture conditions could promote the activity of extracellular peroxidase such as lip and Mn P of Lentinula edodes.Further,primers were designed with the m RNA sequence of Pleurotus eryngii VPL1 obtained from NCBI,and the full-length sequence of VPL1 was obtained by PCR amplification with the c DNA of Pleurotus eryngii 3403 strain as the template,and bioinformatics analysis was carried out.The GPVe P gene expression vector was constructed,and the Lentinula edodes transformants successfully secreting VPL1 protein were obtained by Agrobacterium mediated infection of millet.The results showed that the kinetic constant of recombinant multifunctional peroxidase VPL1 for each substrate was slightly lower than that of strain 3403;It shows significant decolorization effect on dyes with different structure types.The transcription level increased gradually from 12 d to 20 d.In conclusion,this paper obtained the VPL1 gene of Pleurotus eryngii 3403,preliminarily established a rapid and stable detection method for lignin degrading enzyme system in crude enzyme liquid system,the nitrogen limiting medium suitable for the secretion of lip by Lentinula edodes was prepared by plate color development method,constructed the transformation expression system of millet infected by Agrobacterium tumefaciens,successfully obtained the Lentinus edodes transformant secreting active multifunctional peroxidase protein,and studied the properties and functions of VPL1.This paper provides a rapid and simple detection method for Lentinula edodes lignin degrading enzyme system,and also provides a research basis for revealing its expression regulation and the molecular mechanism of lignin degradation.