Functional Verification And Molecular Mechanism Of Laccase In High-temperature Microbial Consortium

Lignin is a complex natural organic polymer and one of the main components of lignocellulose.As the main component of agricultural waste,lignocellulose is the largest renewable biological resource in the world.However,due to the difficulty of lignin degradation,the utilization of straw such agricultural waste resources is limited.A large number of straw is directly burned every year,which causes great environmental pollution.Therefore,it is of great significance to obtain the microbial resources and the functional enzymes which can degrade lignin efficiently for the utilization of straw and environmental protection.In this study,lacZ1 gene fragment encoding laccase was screened from the macro transcriptome data of high temperature lignocellulose degradation microbial consortium WSC-6.RT-q PCR results showed that during the degradation process of lignocellulose,the expression level of lacZ1 gene at the peak of degradation was 30.63 times that of the initial stage of degradation,and the level of expression at the end of degradation stage was 6.07 times that of the initial stage of degradation,and the expression level was significantly increased during the degradation process.The gene was constructed into the cloning plasmid p MD18-T and transformed into competent cell DH5α.The complete nucleic acid sequence of the gene fragment was determined by plasmid sequencing,with the size of 1,548 bp.Phylogenetic tree analysis showed that lacZ1gene encoded laccase and originated from Bacillus in WSC-6.The gene was constructed into the expression plasmid p ET-28a and transformed into the competent cell BL21（DE3）.LacZ1 was purified by nickel column with his tag.The protein size was 59.7 k Da.Resting cells were used to verify the lignin degradation function of lacZ1 gene.It was found that resting cells could degrade90.08%lignin in rice straw within 24 hours.Through the analysis of LacZ1 enzyme properties,it is found that Cu²⁺is an important auxiliary factor of LacZ1.The optimum temperature of LacZ1 is 75°C.when the temperature is not more than 75°C,the laccase activity will not decrease within 1 h,the optimum p H is 4.5,and the maximum enzyme activity can reach 16.39 U/mg.Many common metal ions will inhibit the laccase.Laccase LacZ1 breaks theβ-O-4 bond,β-5 bond,andβ-βbond in lignin,completing the depolymerization of macromolecules.The metabolites of LacZ1 to lignin were determined by GC-MS.nine kinds of mono phenols and lactic acid were found from mass spectrum data.Three degradation pathways of lignin were inferred from the metabolites,namely gentian acid pathway,benzoic acid pathway and protocatechuic acid pathway.This study revealed the molecular mechanism of LacZ1 degrading lignin in WSC-6,which provided a theoretical basis for the degradation of lignin by the high temperature microbial consortium.