Optimization and purification of laccase activity from Mammaliicoccus sciuri isolated from the soils of Similipal, Odisha, India: a kinetics study of crystal violet dye decolorization.

Four laccase-producing bacteria were found in soil samples from the Similipal Biosphere Reserve in Odisha, according to the current study. The isolates (SLCB1 to SLCB4) were evaluated for their laccase-producing ability in LB broth supplemented with guaiacol. The ABTS assay was performed to assess the laccase activity. The bacterium Mammaliicoccus sciuri shows the highest laccase activity i.e., 0.5125 U/L at the optimized conditions of pH 5.5, temperature 32.5 °C, ABTS concentration of 0.75 µl with an incubation time of 9 d. Laccase activity of M. sciuri grown in Sawdust was significantly increased in comparison to that in other agro wastes. The partially purified laccase enzyme after ammonium sulfate precipitation and dialysis showed a molecular weight of ∼58.5 kDa as determined by SDS-PAGE. A decolorization efficiency of 66.67% was recorded for the dye crystal violet after 1 h treatment with dialyzed laccase enzyme compared with phenol red, brilliant blue, and methylene blue.

In silico homology modeling, docking and sequence analysis of some bacterial laccases to unravel enzymatic specificity towards lignin biodegradation.

Laccase is a delignifying enzyme that belongs to the oxidoreductase family, and it has long been investigated as a pretreatment agent in biofuel production. In this study, amino acid sequences of five bacterial laccases from Bifidobacterium breve, Klebsiella pneumonia, Pseudodesulfovibrio hydrargyri, Pseudomonas aeruginosa and Veillonella rodentium have been retrieved from UniProtKB for sequence alignment, phylogenetic analysis using MEGA 7.0 and 3 D structure prediction by homology modeling in SWISS-MODEL. Multiple sequence alignment between all the bacterial laccase sequences revealed a similar structural fold, although the overall protein sequence varied greatly with the substrate binding sites. Further molecular docking in AutoDock Vina and MD stimulation (MDS) in GROMACS for those modelled enzymes were performed considering both apo and ligand bound structures considering both apo and its ligand bound form. Investigation of molecular interaction utilizing docking of five bacterial laccases with three substrates (ABTS, DMP and Guaiacol) revealed that ABTS with K. pneumoniae laccase had the highest binding energy of -7.00 kcal/mol. In the current MDS investigation, bacterial laccases demonstrated greater binding and substrate energy in the ligand bound complex than in the apo form for ABTS, DMP and Guaiacol. In most cases of bacterial laccase, MDS revealed that DMP bound complex was more stable within an average RMSD value lower than 0.5 nm throughout 100 ns time scale. Thus, in silico studies undertaken in this work will be useful in determining the stable enzyme-substrate complex which further might improve the enzymatic catalysis of bacterial laccases for lignin breakdown and biofuel generation.