Bioinformatic Analysis And Enzymatic Characterization Of Myxobacteria And Archaea Laccase-like Multicopper Oxidases

Laccase is a widely used copper-containing oxidase enzyme which can be found in many plants,insects and fungi.Recently,more and more laccases were also found in bacteria.Myxobacteria are an important bacteria resource.However,myxobacteria are much more difficult to isolate and purify than other bacteria.There are few studies on myxobacteria laccase.Archaea are an extreme environment bacteri a and often found to live in extreme environments.These special living habits and the potential of biotechnology development related to this,has been attracting people's attention.We here used BLAST.and Hidden-Markov-Model(HIVMM)methods to screen myxobacteria proteomes available in NCBI.Based on the conserved sequences of four copper binding sites in multicopper oxidase,potential target laccase sequences were obtained.We construct the phylogenetic tree to classify the potential target laccase.Among which,9 genes were chosen by sequence features and categorye s,then synthesized and recombinantly expressed in Escherichia coli BL21(DE3).Seven proteins showed laccase activity when tested with laccase substrates.One protein,namely rSC-2,was chosen for further research because it exhibited the highest activity towards 2,6-dimethyl phenol(DMP).rSC-2 was purified by Ni-NTA affinity chromatography.The optimal temperature and the optimal pH were 60? and 7.0,respectively.About 50%of the original activity was retained after incubation at 60? for 1 h.This is the first report of bioinformatically screening Myxobacteria laccases in combination with expression in E.coli.At the same time,we used the same bioinformatic approach to screen archaea prote omes available in NCBI and 47 potential target laccase sequences were obtained.5 of them were selected for the recombinant expression in E.coli BL21(DE 3)and was no laccase activity.Analysis of the sequence revealed that there are glycosylation sites,two arginine transport pathway and the prediction of the fracture site.It is necessary to post-translational modification and not to obtain the active protein in the E.coli system.The recombinant enzyme was found to be expressed in Pichia pastoris and screened by ABTS.The 5 candidate laccase sequences were expressed in Pichia pastoris(GS115),and the enzyme activity of ABTS was also detected by functional screening.