PURIFICATION AND CHARACTERIZATION OF THERMOSTABLE LACCASE FROM SPOROTHRIX CARNIS

Abstract

Extracellular laccase obtained from Sporothrix carnis was purified in three steps by using ammonium sulphate precipitation, ion-exchange chromatography on a DEAE-Sephacel column, and gel filtration chromatography on Sephadex G-200. The enzyme yield was 3.9%, while the purification fold was 2.84. The purified laccase exhibited optimum activity at 50°C and was able to retain 56% of its original activity after 180 min of incubation at 80°C. The purified enzyme had optimum pH of 7.0 and was stable over a pH range of 3.0 to 11.0. The enzyme activity was enhanced in the presence of Cu2+ and Mn2+ ions, but was reduced by Ca2+, Mg2+, Ba2+ and Hg2+ ions. The purified laccase was mildly inhibited by inhibitors and surfactants and it also showed tolerance for organic solvents. The enzyme oxidized a wide range of substrates that include 2,2’-azino-bis-[3-ethylbenzthiazoline-6-sulfonate] (ABTS), guaiacol, syringaldazine, 4-methylcatechol and pyrocatecol with the highest level of oxidation detected with ABTS. The kinetic parameters Km and VMax of the purified laccase for ABTS were 0.0316 mM and 7.940 mM/min, respectively. The thermostability and other unique characteristics exhibited by laccase from Sporothrix carnis indicate its suitability for application in industrial processes.