Characterization of a Highly Thermostable and Organic Solvent-Tolerant Copper-Containing Polyphenol Oxidase with Dye-Decolorizing Ability from Kurthia huakuii LAM0618T.

Xiang Guo,Weiwei Dong,Guoquan Hu,Mingxiong He,Jinlong Song,Jie Zhu,Zhiyong Ruan,Delong Kong,Yanwei Wang,Huimin Wang,Shan Zhou,Giovanni Maga

doi:10.1371/journal.pone.0164810

Abstract

Laccases are green biocatalysts that possess attractive advantages for the treatment of resistant environmental pollutants and dye effluents. A putative laccase-like gene, laclK, encoding a protein of 29.3 kDa and belonging to the Cu-oxidase_4 superfamily, was cloned and overexpressed in Escherichia coli. The purified recombinant protein LaclK (LaclK) was able to oxidize typical laccase substrates such as 2,6-dimethoxyphenol and l-dopamine. The characteristic adsorption maximums of typical laccases at 330 nm and 610 nm were not detected for LaclK. Cu2+ was essential for substrate oxidation, but the ratio of copper atoms/molecule of LaclK was determined to only be 1:1. Notably, the optimal temperature of LaclK was 85°C with 2,6-dimethoxyphenol as substrates, and the half-life approximately 3 days at 80°C. Furthermore, 10% (v/v) organic solvents (methanol, ethanol, isopropyl alcohol, butyl alcohol, Triton x-100 or dimethyl sulfoxide) could promote enzymatic activity. LaclK exhibited wide-spectrum decolorization ability towards triphenylmethane dyes, azo dyes and aromatic dyes, decolorizing 92% and 94% of Victoria Blue B (25 μM) and Ethyl Violet (25 μM), respectively, at a concentration of 60 U/L after 1 h of incubation at 60°C. Overall, we characterized a novel thermostable and organic solvent-tolerant copper-containing polyphenol oxidase possessing dye-decolorizing ability. These unusual properties make LaclK an alternative for industrial applications, particularly processes that require high-temperature conditions.

Highlights

Laccases are copper-containing polyphenol oxidoreductase enzymes that belong to the multicopper oxidase family [1]
The amino acid sequences of the copper atom binding domains are generally conserved and contain the following four histidine-rich copper binding motifs: HXH, HXHG, HXXHXH and HCHXXXHXXXXM/L/F[3]. Based on their spectroscopic properties, the four copper atoms can be assigned to three types of copper centers: type 1, which is responsible for the blue color of laccases with an absorption maximum of approximately 610 nm; type 2, which is nearly undetectable; and type 3, which has an absorption maximum of approximately 330 nm [2]
Similar sequence searching in BlastP indicated that LaclK is most similar to hypothetical proteins deduced from the genomes of Kurthia massiliensis (WP_010290511) and Kurthia sp

Summary

Introduction

Laccases are copper-containing polyphenol oxidoreductase enzymes that belong to the multicopper oxidase family [1]. The amino acid sequences of the copper atom binding domains are generally conserved and contain the following four histidine-rich copper binding motifs: HXH, HXHG, HXXHXH and HCHXXXHXXXXM/L/F[3]. Based on their spectroscopic properties, the four copper atoms can be assigned to three types of copper centers: type 1, which is responsible for the blue color of laccases with an absorption maximum of approximately 610 nm; type 2, which is nearly undetectable; and type 3, which has an absorption maximum of approximately 330 nm [2]. RL5, a four-copper laccase (28 kDa per monomer) from the bovine rumen, lacks the four characteristic histidine-rich copper binding motifs but exhibits much higher activity than typical laccases [8]

Methods

Results

Discussion

Conclusion