Purification of Laccase from Bacillus licheniformis SO8 with Three-Phase Partitioning, Characterization, and Usage in Dye Decolorization

Laccase is a versatile enzyme that plays an important role in remediation of various environmental pollutants.1 Three-phase partitioning (TPP) is a simple, fast, cost-effective, and highly efficient process that can be used in the purification of laccases.2 In this study, a thermophilic Bacillus licheniformis SO8 (GenBank No: MG076978) was isolated from water samples collected from the Nevşehir Kozaklı hot spring. The laccase was purified to 5.65-fold with 102.07 % recovery using the TPP. The molecular mass of the enzyme was calculated as ~38.7 kDa by SDS-PAGE. Optimum pH 9.0 and optimum temperature for laccase were determined as 70°C. The laccase exhibited pH stability over a wide range (pH 3.0–11.0) and a high thermostability, retaining over 65% of its activity after 1 h of incubation at 20-90°C. It was determined that the enzyme remained highly stable in the presence of 1% and 5% concentrations of surfactants, and increased its activity in the presence of Fe2+ and Mn2+ metal ions. The Km, Vmax, Kcat and Kcat/Km values for the( 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate of the B. licheniformis SO8 laccase were 110 μM, 19.6 μmol.L-1.dk-1, 0.048 s-1 and 0.44 s-1.mM-1, respectively. Finally, Acid black 1, Congo red, Methylene blue, Orange, Acid red 27, Reactive black 5 dyes were used to examine the effect of laccase on the color removal of some synthetic dyes and it was determined that the highest dye removal was on Acid Red 27 dye with ~38%. In conclusion, the remarkable properties of the laccase enzyme isolated from B. licheniformis SO8 may offer an important opportunity to degrade environmental pollutants, making it an attractive biocatalyst for industrial applications.