Recombinant CotA-Laccase from Bacillus Licheniformis: Characterization and Overexpression in Bacillus Subtilis WB۶۰۰

Background: Laccases are a class of multi-copper oxidases that can catalyze the oxidation of various phenolic substrates while reduacing molecular oxygen to water. Although only a few bacterial laccases have been studied to date, recent advances in genome research suggest that these enzymes are widespread in bacteria. Due to their ability to oxidize a broad range of phenolic compounds, laccases have numerous biotechnological applications. The aim of this study was to isolate the gene encoding laccase (CotA) from recombinant Escherichia coli BL۲۱ (DE۳) containing the Bacillus licheniformis LS۰۴ CotA-laccase gene and investigate its properties.Methods: The bacterial strains, vectors, and growth conditions were used in the study, and also the recombinant and expression host strain construction was described. Plasmid isolation, PCR amplification, gel electrophoresis, and protein purification were also carried out. SDS-PAGE was used to visualize the protein bands and plasmid stability was analyzed. In addition, this study characterized the CotA laccase by evaluating its optimum temperature, pH, thermal stability, and activity after bathing at ۵۰ °C for ۱۰ min.Results: The results showed that the CotA laccase produced a protein with a molecular weight of ۶۵ kDa, and the plasmid was stable in the absence of antibiotic pressure for ۲۰۰ generations. The pH profile for laccase activity showed a peak at pH ۷.۴, and the optimal temperature was found to be ۴۵ °C. However, the pH and temperature stability of the CotA laccase was lower than that of the spore laccase.Conclusion: The purified recombinant CotA-laccase showed high stability towards alkaline pH, high temperatures, and a broad pH range for catalyzing substrates. Nevertheless, the study demonstrates that CotA-laccase has the potential for industrial use due to its high stability and broad substrate range.

کلیدواژه ها:

Bacillus subtilis ، Bacillus licheniformis ، Cloning ، CotA ، Laccase