Expression,Purification,Characterization And Application Of A Novel Bacterial Laccase Melac13220

Laccase is a blue copper oxidase.It widely exists in fungi,plants,insects and bacteria.Laccase has a broad substrate specificity and can oxidize a variety of phenolic and non-phenolic aromatic compounds.Bacterial laccases are different from fungal laccases in their ability to produce intracellular or extracellular laccases.They are active within a wide range of temperatures and p H ranges.Bacterial laccases can be used for pulp biobleaching,bioremediation,decolorization of textile dyes,pollutant degradation and biosensors,etc.Therefore,comprehensive information including sources,production conditions,cloning,characterization,and biotechnology applications needs to be analyzed and compared in order to effectively understand and apply these enzymes at the industrial level.In this study,the laccase Melac gene was amplified from the genome of Methylobacterium extorquens.The expression vector was successfully constructed and expressed highly in E.coli with a molecular weight of approximately 50 k Da.It was purified by ion exchange chromatography.The purity of the enzyme after purification is high and the enzyme can be directly used for subsequent characterization and application studies.The experimental results show that the optimum temperature of the enzyme was 75°C when ABTS was used as the substrate to determine the enzyme activity,but the stability of the enzyme was poor when the temperature was over 50°C.When the temperature was 40°C,it shows the best stability with the half-life of 50 hours and the activity declines slowly.The half-life at 45° C is about 40 hours.The half-life is 8.5 h at 50° C,and the enzyme activity declines rapidly.The optimum p H of this enzyme is 1.5,and the stability under acidic conditions is good.After 1 hour,the enzyme can still remain more than 80% of its activity.The Km value for the substrate ABTS is 7.65 x 10-2 m M and the kcat value is 14.6/s.Cu2+ and Co2+ can slightly increase enzyme activity,while Fe2+ can increase enzyme activity by 25-fold,probably because Fe2+ affects the class I copper ion site at the active center of the enzyme.Different organic reagents will reduce enzyme activity to varying degrees.In the absence of any mediator,the decolorization efficiency of Congo Red by this laccase is higher,and the decolorization rate is more than 90% at 10 h.The decolorization efficiency is low for other dyes such as indigo carmine.This study mainly included the cloning,expression,purification,properties and applications of a novel bacterial laccase from Methylobacterium extorquens.This study discovered the differences between this enzyme and other laccases(such as the effect of Fe2+ on enzyme activity).It also has provided experimental basis for in-depth study of the enzyme,hoping that it can be more valuable in applications by changing the properties of the new enzyme.