Heterologous expression of bacterial CotA-laccase, characterization and its application for biodegradation of malachite green

Malachite green (MG) is used as fungicide/parasiticide in aquaculture, its persistence is detrimental as it exhibits carcinogenic effects to aquatic organisms. Bacterial laccase evaluated as the best enzyme at extreme condition for aquatic MG removal. Study aims to increase laccase concentration, CotA-laccase from Bacillus subtilis was cloned and overexpressed in Escherichia coli. Optimal catalysis for purified CotA-laccase were at pH 5.0, 60 degrees C, and 1 mM of (2,2-azino-di-[3-ethylbenzothiazoline-sulphonate-(6)]) with Km and Kcat 0.087 mM and 37.64 S-1 respectively. MG biodegradation by CotA-laccase in clam and tilapia pond wastewaters and cytotoxic effect of biodegraded products in grouper fin-1 cells were determined. MG degradation by CotA-laccase was equally efficient, exhibiting upto 90-94% decolorization at freshwater and saline conditions and treated solution was non-toxic to GF-1 cells. Thus, recombinant-CotA-laccase could be an environmentally-friendly enzyme for aquaculture to remove MG, thereby effective to reduce its accumulation in aquatic organisms and ensuring safe aquaculture products.

Automated summary

Bacterial laccase was found to be the best enzyme for removing malachite green in aquaculture, with cloned and overexpressed CotA-laccase showing high efficiency in degrading MG in both freshwater and saline conditions. The biodegraded products of MG by CotA-laccase had minimal cytotoxic effects on grouper fin-1 cells.