Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in the degradation of pharmaceuticals

Abstract

Recent years have seen an increasing interest in laccase enzymes. Due to their ability of oxidizing various substrates, they are nowadays applied in multiple industrial fields including pulp delignification, textile dye bleaching, and bioremediation. In contrast to laccase production from native sources, with its generally low yield and high cost, heterologous laccase expression is far better suited to meet the growing industrial demands. TVLCC5 gene encoding Trametes versicolor laccase 5 was overexpressed in Arxula adeninivorans using the strong constitutive TEF1 promoter. Recombinant Tvlcc5 protein was purified by immobilized-metal ion affinity chromatography and biochemically characterized using 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as substrate for standard activity assays. The enzyme showed the highest activity at 50\xa0°C between pH 4.5–5.5. The half-life of Tvlcc5 at 60\xa0°C was around 20\xa0min. The negative effect of chloride anions on enzyme activity was demonstrated. A fed-batch cultivation of Tvlcc5 producing strain A. adeninivorans G1212/YRC102-TEF1-TVLCC5-6H was performed and resulted in a laccase activity of 4986.3\xa0U\xa0L−1. To improve the expression level of recombinant laccase in A. adeninivorans, cultivation conditions were optimized by single factor experiments. Recombinant Tvlcc5 proved to be a promising agent for degradation of pharmaceuticals that are an important source of environmental pollution. Concentration of diclofenac and sulfamethoxazole decreased to 46.8% and 51.1% respectively after 24\xa0h incubation with Tvlcc5. When 1\xa0mM redox mediator ABTS was added complete degradation was obtained within 1\xa0h.