Comparative Studies on Recombinant Laccases of Thermiphilic Geobacillus Sbs-4S and Mesophilic Bacillus Strain R5 Origins

Laccases are multidomain copper containing proteins acting on phenolic and non-phenolic compounds. They are industrially relevant enzymes performing diverse oxidative functions including dye decolorization, food processing, organic synthesis and bio-remediation. Laccases exist in all domains of life. Laccases from fungal species are well characterized for industrial utilization. However, Laccases from bacterial genera are comparatively less characterized. In comparison with fungal laccases bacterial laccases are heat stable and halide tolerant, the properties desired for industrial applications. The present study describes the identification, cloning, gene expression and characterization of laccases from two sources (i) from thermophilic Geobacillus thermopakistaniensis and (ii) from mesophilic Bacillus subtilis strain R5. Genome search of Geobacillus thermopakistaniensis, formerly Geobacillus sp. SBS-4S, revealed the presence of an open reading frame (GenBank accession number ESU71923) annotated as laccase, (named as Gt-Lac). Gt-Lac gene was having 825 nucleotides, encoding a protein of 273 amino acids. The BLAST search showed that Gt-Lac does not display sequence similarity with characterized laccases of Bacillus subtilis, Streptomyces coelicolor and Thermus thermophilus. Gt-Lac shared highest homology with laccases of a new protein family, DUF152, like Kurthia huakuii (32%), RL5 laccase from bovine rumen metagenome (31%), and Thermobifida fusca (28%). To examine the properties of ESU71923, Gt-Lac was cloned in expression vector (pET-21a) and mobilized to E. coli for the production of recombinant enzyme. However, the purified recombinant protein did not exhibit any laccase-like activity even when produced in the presence of copper ions. Expression of Gt-Lac gene at low temperatures and in the presence of zinc also failed to produce an active enzyme. Atomic absorption spectroscopy could detect only zinc atom instead of four coppers that most laccases possess. Based on these results it was suggested that ESU71923 does not encode a functional laccase. The laccase activity was, therefore, purified from G. thermopakistaniensis cells and N-terminal amino acid residues of the enzyme were determined. These residues matched the N-terminal sequence of an open reading frame annotated as a copper oxidase (ESU72270). In order to characterize the enzyme, recombinant ESU72270 (named as Gt-Cuo) was prepared in Escherichia coli. Gt-Cuo gene encoded a protein of 503 amino acids with a molecular weight of about 60 kDa. The recombinant protein was found to exhibit a negligible amount of laccase activity when produced in the absence of copper in the growth medium. However, the recombinant protein exhibited significantly high laccase activity when produced in the presence of copper. The recombinant enzyme showed highest activity at 60 °C and a pH of 7–7.5. Gt-Cuo was copper dependent and a five-fold increase in laccase activity was observed in the presence of 100 μM copper sulfate. When using halide donors, a 7-fold and 5-fold increase in laccase activity was observed with 500 mM NaBr and NaCl, respectively. Gt-Cuo showed good stability towards organic solvents. Moreover Gt-Cuo was able to decolorize several synthetic dyes with highest rate of color removal observed for indigo caramine. In conclusion, this is the first report about the identification of gene, from genus Geobacillus, responsible for true laccase activity having potential to be used for biotechnological applications. The third gene of the study, (named as Bsu-Lac) laccase from Bacillus subtilis was composed of 513 amino acids with calculated molecular weight of 58498.99 Da. When expressed in E. coli the recombinant Bsu-Lac was produced as inclusion bodies which were tried to refold by denaturing in urea but the refolded sample was inactive. Co-expression of Bsu-Lac gene with a chaperonin gene also failed to solubilize the inclusion bodies. Finally expression of Bsu-Lac gene was taken in pET-28a(+) at low temperature and the protein was purified by nickel affinity chromatography. The enzyme was found to function optimally at 55 °C and a pH of 7. The laccase activity of Bsu-Lac was dependant on copper only during protein production rather than adding in assay mixture, showing the importance of copper for proper protein folding. The enzyme was stable in 10% of all tested organic solvents. The Bsu-Lac activity was stable at 80 °C for 150 min. When the protein was incubated with various concentrations of urea, structural stability was found even at 8 M urea. The recombinant protein was able to degrade various synthetic dyes with the highest rate of dye degradation for orange G, thus having potential for various industrial applications.