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

Felix Bischoff,Katarzyna Litwińska,Falko Matthes,Twan Rutten,Rüdiger Bode,Gotthard Kunze

doi:10.1186/s13568-019-0832-3

Felix Bischoff, Katarzyna Litwińska + Show 4 more

Open Access

https://doi.org/10.1186/s13568-019-0832-3

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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 °C between pH 4.5–5.5. The half-life of Tvlcc5 at 60 °C was around 20 min. 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 U L−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 h incubation with Tvlcc5. When 1 mM redox mediator ABTS was added complete degradation was obtained within 1 h.

Highlights

Annual worldwide intake of active pharmaceutical ingredients (APIs) is estimated at 100,000 tons (Touraud and Roig 2008)
Successful gene insertion (Additional file 1: Fig. S1) as well as transcription were confirmed for all laccase genes (Additional file 1: Fig. S3), no other than TVLCC5 leads to detectable protein neither in supernatant nor within the cells
0.0 ± 4.9 106.0 ± 0.9 108.9 ± 1.6 99.3 ± 3.0 104.9 ± 0.7 116.6 ± 2.3 100.0 ± 2.3 were already detectable after 2 h of cultivation, whereas signals for mRNA of TVLCC5 and TVLCC2 were first found in cDNA from samples taken after 24 h

Summary

Introduction

Annual worldwide intake of active pharmaceutical ingredients (APIs) is estimated at 100,000 tons (Touraud and Roig 2008). Due to the low absorption capacity of most APIs, they leave the human body unaffected or as a metabolite over the renal system, ending up in the world’s water system. This problem is intensified through improper disposal of unused or expired medicinal products. In EU 50% or more of unused pharmaceuticals is not returned to the pharmacy (European Environment Agency 2010; BIO Intelligence Service 2013). The amount of unused medicinal products in Germany is assessed to 5700 tons/year.

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