Abstract
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 °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. 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.
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Data Availability
The 16s rRNA sequence of the Bacillus aerius SP3 was deposited in GenBank with accession number MZ404271.1 and available in the URL https://www.ncbi.nlm.nih.gov/nuccore/MZ404271.1. The results of OFAT optimization is provided as supplementary material.
Abbreviations
- OFAT:
-
One-factor-at-a-time
- RSM:
-
Response surface methodology
- BBD:
-
Box–Behnken design
- DEAE:
-
Diethyl aminoethyl
- ABTS:
-
2,2-Azino-bis-3-ethyl benzothiazoline-6-sulphonic acid
- DMP:
-
Dimethoxy phenol
- MSM:
-
Mineral salt medium
- ANOVA:
-
Analysis of variance
- HOBT:
-
Hydroxybenzotriazole
- CV:
-
Crystal violet
- MB:
-
Methylene blue
- CVM:
-
Crystal violet with mediator
- MBM:
-
Methylene blue with mediator
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The authors acknowledge the Department of Industrial Biotechnology and Environmental Engineering of Government College of Technology, Coimbatore, for granting permission to avail the instrumentation facilities to carry out the work.
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Saranya, P., Jayanthi, S. & Nagappan, S. Production and characterization of extracellular laccase from Bacillus aerius SP3 and its application in dye decolorization. Biologia 79, 1497–1511 (2024). https://doi.org/10.1007/s11756-024-01619-3
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DOI: https://doi.org/10.1007/s11756-024-01619-3