Production and characterization of extracellular laccase from Bacillus aerius SP3 and its application in dye decolorization

Laccases are lignolytic enzymes that catalyze and cleave various environmental pollutants. In this study, we isolated a novel laccase-producing bacterium, Bacillus aerius SP3, from compost soil using ABTS as the substrate. The production of extracellular laccase was improved through the one-factor-at-a-time (OFAT) method and response surface methodology using the Box-Behnken design (BBD) in submerged fermentation. Optimization of medium components, including 20 g/L starch, 10 g/L peptone, 3.36 mM CuSO4, and 72-h incubation time, resulted in a 3.46-fold increase in extracellular laccase production compared with the unoptimized medium. The extracellular laccase obtained from the culture supernatant was purified through 60% ammonium sulfate precipitation, dialysis, and ion exchange chromatography using a DEAE cellulose column. SDS-PAGE estimated the molecular weight of the isolated laccase to be approximately 60 kDa. The purified laccase also decolorized textile dyes, crystal violet and methylene blue, achieving 48% and 22% removal in the presence of ABTS as a mediator. The optimal temperature and pH for laccase activity were 35( degrees)C and 7.0, respectively. The enzyme demonstrated stability up to 40 C-degrees for 60 min and at pH 8.0 at 4 C-degrees for 24 h. Cu2+ and Zn2+ enhanced laccase activity by 110%, while > 50% of activity was retained in the presence of heavy metals such as Hg2+ and Cd2+. Organic solvents, including 10% ethanol and methanol, increased laccase activity by 1.5 times. These results indicate that the laccase is active at neutral/alkaline pH, tolerant to heavy metals, and organic solvents. Enhancement of laccase activity by organic solvents is advantageous for chemical reactions in solvent environments and treating wastewater from dyeing industries.