Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense.

Wissal Ben Ali,Annick Turbé-Doan,Eric Record,Tahar Mechichi,Yann Mathieu,Amal Ben Ayed,Craig B Faulds,Emmanuel Bertrand,Anne Lomascolo,Giuliano Sciara

doi:10.3390/ijms21218402

Abstract

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L−1. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.

Highlights

The laccases belong to a small group of enzymes called the blue copper protein or copper oxidases [1] that catalyse the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water [2]
Laccase-catalysed reactions are strongly dependent on redox potential, temperature and reaction medium [11]
Low-redox-potential enzymes occur in bacteria and plants, and high-redox-potential laccases are widely distributed in fungi [12,13]

Summary

Introduction

The laccases (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) belong to a small group of enzymes called the blue copper protein or copper oxidases [1] that catalyse the one-electron oxidation of four reducing-substrate molecules concomitant with the four-electron reduction of molecular oxygen to water [2]. Laccases are almost ubiquitous enzymes, widely distributed among plants, fungi, prokaryotes, and arthropods [3]. Laccases have active sites containing four copper atoms (Cu) bound to three redox sites (Type 1, Type 2, and Type 3 Cu pair) involved in the catalytic mechanisms of these cuproproteins [7,8]. Type 1 Cu at its oxidised resting state is responsible for the blue colour of the protein and is EPR-detectable. Laccases exhibit broad substrate ranges that vary from one laccase to another [10] They are known as p-diphenol:oxygen oxidoreductases, preferentially oxidising monophenols such as 2,6-dimethoxyphenol or guaiacol. Low-redox-potential enzymes occur in bacteria and plants, and high-redox-potential laccases are widely distributed in fungi [12,13]. Many industrial applications for laccases have been proposed, including in pulp and paper [17], organic synthesis, environment, food, pharmaceuticals, and textile dye decolourisation [18]

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