@phdthesis{Litwińska2020, author = {Katarzyna Litwińska}, title = {Characterization of recombinant laccase from Trametes versicolor synthesized by Arxula adeninivorans and its application in enzymatic removal of pharmaceuticals from wastewater}, journal = {Charakterisierung einer durch Arxula adeninivorans synthetisierten, rekombinanten Laccase von Trametes versicolor und deren Anwendung im enzymatischen Abbau von Pharmazeutika in Abwasser}, url = {https://nbn-resolving.org/urn:nbn:de:gbv:9-opus-38731}, pages = {140}, year = {2020}, abstract = {In recent years, negative impact of pharmaceutical products on natural environment became an issue of high public interest. Pharmaceutical residues are detected in various ecosystems worldwide. Due to increasing production and consumption of medicines this problem is intensified. Therefore, an efficient way to restrain release into the world’s water system is required. This work presents an enzymatic approach for the degradation of pharmaceuticals in wastewater treatment plants, using laccase and cytochrome P450 — two enzymes of high biotechnological and industrial potential. Laccase genes from fungi Trametes versicolor and Pycnoporus cinnabarinus were isolated and overexpressed in the non-conventional yeast Arxula adeninivorans. This organism served also as cytochrome P450 gene donor. Recombinant laccase Tvlcc5 was purified by immobilized-metal ion affinity chromatography and biochemically characterized using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as substrate for enzyme activity assays. The optimal temperature and pH were found to be 50 °C and 4.5–5.5, respectively. The half-life of Tvlcc5 at 60 °C was around 20 min. It was demonstrated that the presence of copper ions is essential for the synthesis of active protein. Moreover, negative impact of chloride anions on laccase activity was shown. Cultivation conditions for the Tvlcc5 producing strain A. adeninivorans G1212/YRC102-TEF1-TVLCC5-6H were optimized. It was found that maintaining the pH at a constant level between pH 6.0 and 7.0 is essential for the production of active enzyme. Optimal cell growth and laccase accumulation were reached at 20 °C and in medium supplemented with 0.5 mM CuSO4. Performed fed-batch cultivation resulted in a laccase activity of 4986.3 U L-1. Factors influencing the synthesis of Tvlcc5 leading to increased production of this protein were investigated. It was found that using three non-native signal peptides (cutinase 2 from A. adeninivorans (ACut2), α-mating factor from S. cerevisiae (MFα), and acid phosphatase from P. pastoris (PHO1) signal peptides) enhances the secretion of active enzyme by 20–80\%. Besides that, additional overexpression of copper transporters positively affects laccase production. Finally, it was proven that recombinant Tvlcc5 is a promising agent for the degradation of certain pharmaceuticals. After 24 h of incubation, the concentration of diclofenac and sulfamethoxazole decreased to 46.8\% and 51.1\%, respectively. Furthermore, it was shown that the addition of the redox mediator ABTS significantly shortens the degradation time of these substances.}, language = {en} }