Screening Of Lignin Degrading Bacteria And Cloning And Analysis Of Lignin Peroxidase Gene

As the second largest renewable raw material in nature,the efficient and clean degradation and utilization of lignin are urgently needed.Compared with physical and chemical methods,microbial degradation methods have the most advantages due to their specificity and environmental friendliness.Lignin peroxidase is a key enzyme for lignin degradation.Therefore the study on lignin peroxidase gene is of great significance for the research,development and utilization of lignin peroxidase,as well as for the regulation of lignin degradation of microorganisms.In this paper,a strain of lignin-degrading bacteria M1 was selected from the rotten wood soil,which was identified as Acinetobacter by physiological and biochemical experiments and 16S rDNA.The lignin-producing peroxidase of M1 was optimized by controlling a single variable.The results showed that when M1 was at 36?,the p H value of medium was 7,the rotation speed was 160 r/min,the carbon source was sucrose,and the nitrogen source was peptone,the enzyme production conditions were the best,and the enzyme activity reached68.15 U/L after continuous culture for 24 h.The target gene sequence of 829 bp was cloned from M1 genomic DNA,encoding 270amino acids.The molecular formula C₁₄₁₃H₂₁₅₁N₃₉₁O₃₈₅S₁₄,the molecular weight was31224.85Da,and the isoelectricpoint size was 8.39.The sequence comparison showed that it was 70%similar to the Trametes versicolor FP-101664 SS1 lignin peroxidase gene(sequence ID:NW 007360332.1).At the same time,the sequence also contains benzohis/livelihood hydrolase,which has been shown to catalyze the cleavage of ester bonds between lignin and glucuronic acid groups,and thus complete the degradation of lignin by bacteria.It contains the core binding region of Nudix Hydrolase superfamily,which can catalyze the generation of the cofactor pr FMN necessary for the biodegradation of lignin and the biotransformation of aromatic compounds.p ET32a(+)was used to construct the expression vector p ET32a(+)-lip,which was transformed into Escherichia coli BL21(DE)competent cell.The target protein was induced and expressed by IPTG at a concentration of 1.0 mmol/L.The expressed fusion protein had a molecular weight of 31.2k Da.The discovery of M1 provides a new choice of strain for microbial degradation of lignin,and the optimization of conditions for producing lignin peroxidase can provide a theoretical basis for its industrial application.Meanwhile,the cloning and analysis of its lignin peroxidase gene can further promote the development of microbial degradation of lignin.