Expression of a new laccase from Moniliophthora roreri at high levels in Pichia pastoris and its potential application in micropollutant degradation

Peter-Leon Hagedoorn,Vlada B Urlacher,Agathe Bronikowski,Katja Koschorreck

doi:10.1186/s13568-017-0368-3

Peter-Leon Hagedoorn, Vlada B Urlacher + Show 2 more

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https://doi.org/10.1186/s13568-017-0368-3

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Abstract

Laccases have gained significant attention due to their emerging applications including bioremediation, biomass degradation and biofuel cells. One of the prerequisites for the industrial application of laccases is their sufficient availability. However, expression levels of recombinantly expressed laccases are often low. In this study Mrl2, a new laccase from the basidiomycete Moniliophthora roreri, was cloned in Pichia pastoris and produced in an optimized fed-batch process at an exceptionally high yield of 1.05 g l−1. With a redox potential of 0.58 V, Mrl2 belongs to mid-redox potential laccases. However, Mrl2 demonstrated high kcat values of 316, 20, 74, and 36 s−1 towards 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), syringaldazine (SGZ), 2,6-dimethoxyphenol (2,6-DMP) and guaiacol, respectively. Mrl2 remained stable above pH 6 and in the presence of many metal ions, which is important for application in bioremediation. Mrl2 was investigated for the ability to degrade endocrine disrupting chemicals (EDCs) and non-steroidal anti-inflammatory drugs (NSDAIs) at neutral pH value. The enzyme accepted and converted estrone, 17β-estradiol, estriol, the synthetic contraceptive 17α-ethinyl estradiol and bisphenol A at pH 7 faster than high-potential laccases from Trametes versicolor. For example, within 30 min Mrl2 removed more than 90% bisphenol A, 17ß-estradiol, 17α-ethinyl estradiol and estriol, respectively. The concentration of the recalcitrant drug diclofenac dropped by 56% after 20 h incubation with Mrl2.

Highlights

Laccases (EC 1.10.3.2) are multi-copper oxidases which catalyze the oxidation of various electron-rich organic and inorganic molecules like mono- and diphenols, polyphenols, diamines, aminophenols, aromatic or aliphatic amines with the four-electron reduction of molecular oxygen to water (Thurston 1994; Xu 1996; Xu et al 1996)
Identification of a putative high‐potential laccase The catalytic activity of laccases is influenced by the difference in the redox potential of a substrate and the type 1 copper (Xu 1996; Xu et al 1996)
To identify a laccase with a high redox potential, a BLAST search was done with the motif HCHIDWHLEAGF, containing the coordinating histidines of the type 1 (T1) and T3 copper ions, the LEA tripeptide and the non-coordinating axial ligand phenylalanine, that is highly conserved within high-potential laccases (Mot and Silaghi-Dumitrescu 2012)

Summary

Introduction

Laccases (EC 1.10.3.2) are multi-copper oxidases which catalyze the oxidation of various electron-rich organic and inorganic molecules like mono- and diphenols, polyphenols, diamines, aminophenols, aromatic or aliphatic amines with the four-electron reduction of molecular oxygen to water (Thurston 1994; Xu 1996; Xu et al 1996). High-potential laccases typically possess a broader substrate spectrum than low- or middle-potential laccases and are even able to oxidize substrates with a redox potential of up to 1.2 or 1.4 V (Tadesse et al 2008). Regarding their broad substrate spectrum while only relying on water as a cosubstrate, laccases often are called “green” biocatalysts (Pardo and Camarero 2015). These enzymes, and highpotential laccases in particular, are attractive for industrial applications as in the pulp and paper, food and textile industry, nanobiotechnology, synthetic chemistry or bioremediation (Rodriguez Couto and Toca Herrera 2006)

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