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

• Published in: Biológia Vol. 79; no. 5; pp. 1497 - 1511
• Main Authors: Saranya, Palanisamy, Jayanthi, Singaram, Nagappan, Senthil
• Format: Journal Article
• Language: English
• Published: Cham Springer International Publishing 01.05.2024; Springer Nature B.V
• Subjects: Ammonium; Ammonium sulfate; Bacillus aerius; Biomedical and Life Sciences; Cell Biology; Cell culture; Cellulose; Chemical reactions; Color removal; Decoloring; Decolorization; Design optimization; Dialysis; Dyeing; Dyes; Ethanol; Fermentation; Gentian violet; Heavy metals; Ion exchange; Laccase; Life Sciences; Ligninolytic enzymes; Methylene blue; Microbiology; Molecular weight; Organic solvents; Original Article; Peptones; pH effects; Plant Sciences; Response surface methodology; Solvents; Substrates; Wastewater treatment; Zinc; Zoology; Bacterial laccase; Screening; Box–Behnken design; RSM; ABTS
• ISSN: 1336-9563; 0006-3088; 1336-9563
• DOI: 10.1007/s11756-024-01619-3

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 CuSO 4 , 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 °C and 7.0, respectively. The enzyme demonstrated stability up to 40 °C for 60 min and at pH 8.0 at 4 °C for 24 h. Cu 2+ and Zn 2+ enhanced laccase activity by 110%, while > 50% of activity was retained in the presence of heavy metals such as Hg 2+ and Cd 2+ . 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.