Purification and biochemical characterization of a new thermostable laccase from Enterococcus faecium A2 by a three-phase partitioning method and investigation of its decolorization potential.

Three-phase partitioning (TPP) is a simple, fast, cost-effective, and highly efficient process that can be used in the purification of laccases. In this study, microorganisms with laccase activity were isolated from water samples collected from the Agri-Diyadin hot spring. The isolate with the highest laccase activity was found to be the A2 strain. As a result of molecular (16S rRNA sequence) and conventional (morphological, biochemical, and physiological) analyses, it was determined that the A2 isolate was 99% similar to Enterococcus faecium (Genbank number: MH424896). The laccase was purified to 4.9-fold with 110% recovery using the TPP. The molecular mass of the enzyme was found by SDS-PAGE to be 50.11 kDa. Optimum pH 6.0 and optimum temperature for laccase were determined as 80 °C. The laccase exhibited pH stability over a wide range (pH 3.0-9.0) and a high thermostability, retaining over 90% of its activity after 1 h of incubation at 20-90 °C. The laccase exhibited high thermostability, with a heat inactivation half-life of approximately 24 h at 80 °C. The enzyme remained highly stable in the presence of surfactants and increased its activity in the presence of organic solvents, Cr, Cu, and Ag metal ions. The K, V, k, and k/K values of laccase for 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) substrate were 0.68 mM, 5.29 μmol mL min, 110.2 s, and 162.1 s mM, respectively.