Cohnella sp. A01 laccase: thermostable, detergent resistant, anti-environmental and industrial pollutants enzyme

Masoomeh Shafiei,Farzaneh Afzali,Ali Asghar Karkhane,S Mehdi Ebrahimi,Kamahldin Haghbeen,Saeed Aminzadeh

doi:10.1016/j.heliyon.2019.e02543

Masoomeh Shafiei, Farzaneh Afzali + Show 4 more

Open Access

https://doi.org/10.1016/j.heliyon.2019.e02543

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Abstract

Laccase (EC 1.10.3.2; benzenediol; oxygen oxidoreductases) is a multi-copper oxidase that catalyzes the oxidation of phenols, polyphenols, aromatic amines, and different non-phenolic substrates with concomitant reduction of O2 to H2O. Enzymatic oxidation techniques have the potential of implementation in different areas of industrial fields. In this study, the Cohnella sp. A01 laccase gene was cloned into pET-26 (b+) vector and was transformed to E. coli BL21. Then it was purified using His tag affinity (Ni sepharose resin) chromatography. The estimated molecular weight was approximately 60 kDa using SDS-PAGE. The highest enzyme activity and best pH for 2,6-dimethoxyphenol (DMP) oxidation were recorded as 8 at 90 °C respectively. The calculated half-life and kinetic values including Km, Vmax, turn over number (kcat), and catalytic efficiency (kcat/Km) of the enzyme were 106 min at 90 °C and 686 μM, 10.69 U/ml, 20.3 S−, and 0.029 s−1 μM−1, respectively. The DMP was available as the substrate in all the calculations. Enzyme activity enhanced in the presence of Cu2+, NaCl, SDS, n-hexane, Triton X-100, tween 20, and tween 80, significantly. The binding residues were predicted and mapped upon the modeled tertiary structure of identified laccase. The remaining activity and structural properties of Cohnella sp. A01 laccase in extreme conditions such as high temperatures and presence of metals, detergents, and organic solvents suggest the potential of this enzyme in biotechnological and industrial applications. This process has been patented in Iranian Intellectual Property Centre under License No: 91325.

Highlights

Laccase (benzodiol; oxygen oxidoreductase (EC.1.10.3.2)) is a multicopper oxidase catalyzing the oxidation of phenols, poly-phenols, aromatic amines, and a variety of non-phenolic substrates [1]
A01 laccase was exogenously expressed in bacterial host, purified, and characterized for its biochemical, thermodynamic, and structural features in the present study
This enzyme was naturally resistant to high temperatures, a wide pH range, and the presence of ions, surfactants, and organic solvents

Summary

Introduction

Laccase (benzodiol; oxygen oxidoreductase (EC.1.10.3.2)) is a multicopper oxidase catalyzing the oxidation of phenols, poly-phenols, aromatic amines, and a variety of non-phenolic substrates [1]. The oxygen molecule (O2) first oxidizes the enzyme to produce the reactive intermediate (laccase-oxygen) along water; laccase-oxygen removes one electron from the substrate [2]. This highly reactive substrate-driven radical is able to participate in a variety of reactions such as rearrangements and polymerizations. The initial laccase will be reformed through obtaining four dismissed electrons during oxygen reduction [3]. The type I copper has been suggested to have initial substrate oxidation role and its absence leads to enzyme inactivation. The type III contains two copper atoms that are responsible for oxygen reduction. The reduction of type I copper by an organic compound is the prerequisite of the oxygen attachment to type II/III coppers [3]

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