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

Abstract

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, Km and Vmax 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.