Purification of laccase from thermophilic Bacillus licheniformis SO8 with three-phase partitioning, characterization, and usage in dye decolorization

Laccase is an environmentally friendly biocatalyst capable of oxidizing a wide range of organic compounds and has attracted considerable interest for industrial wastewater treatment applications. In this study, laccase from the thermophilic Bacillus licheniformis SO8 strain (GenBank no: MG076978) was purified and characterized using the three-phase partitioning (TPP) method, a simple, rapid, low-cost, and efficient bioseparation technique, and its dye decolorization capability was preliminarily evaluated. The enzyme was purified 5.65-fold with a recovery of 102.07% under optimal TPP conditions (pH 9.0, t-butanol ratio of 1.0:1.0, and 70% ammonium sulfate), and its molecular weight was determined to be about 38.7 kDa. The purified laccase exhibited optimal activity at pH 9.0 and 70 °C and retained approximately 65% of its activity over a broad pH and temperature range. Enzyme activity was enhanced in the presence of Fe²⁺ and Mn²⁺ ions. The kinetic parameters for 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were determined as K m 110 µM, V max 19.6 µmol L⁻¹ min⁻¹, k cat 0.048 s⁻¹, and k cat /Km 0.44 s⁻¹ mM⁻¹. Dye decolorization experiments indicated moderate removal efficiency, with a maximum of 38% observed for Acid Red 27, suggesting that the thermophilic laccase may serve as a potential biocatalyst for dye treatment under optimized conditions.