Biochemical characterization of an extremely stable pH-versatile laccase from Sporothrix carnis CPF-05

Functionality of enzymes within narrow pH range and temperature is a major challenge which limits their industrial applications, hence, there is need to search for thermostable pH-versatile enzymes. Here, a novel thermostable pH-versatile laccase from Sporothrix carnis CPF-05 was purified by ion-exchange and gel filtration chromatography. Single protein band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) confirmed homogeneity of the enzyme with molecular weight of 56kDa. Enzyme yield was 3.9% and purification fold was 2.84. Purified laccase exhibited optimum activity at 50°C and retained 56% of its initial activity at 80°C after 180min of incubation with 2,2' azino-di-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate. The enzyme had optimum pH of 7.0 and was stable over pH range of 3.0 to 11.0. Laccase activity was enhanced by Cu²⁺ and Mn²⁺ ions but inhibited by Ca²⁺, Mg²⁺, Ba²⁺and Hg²⁺ ions. Purified laccase was mildly inhibited by urea, sodium azide and surfactants while exhibiting tolerance to organic solvents. The enzyme demonstrated broad substrate specificity. Kinetic parameters, K_m and V_max of the purified laccase for ABTS were 0.0316mM and 7.940mM/min, respectively. Thermostability, pH-versatility and other characteristics of laccase from S. carnis CPF-05 indicate its suitability for variety of industrial processes.