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

PLoS One. 2016 Oct 14;11(10):e0164810. doi: 10.1371/journal.pone.0164810. eCollection 2016.

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.

MeSH terms

  • Amino Acid Sequence
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Biocatalysis
  • Catechol Oxidase / genetics
  • Catechol Oxidase / metabolism*
  • Cloning, Molecular
  • Coloring Agents / chemistry
  • Coloring Agents / metabolism*
  • Copper / chemistry*
  • Dopamine / metabolism
  • Escherichia coli / metabolism
  • Firmicutes / metabolism*
  • Hydrogen-Ion Concentration
  • Kinetics
  • Molecular Sequence Data
  • Oxidation-Reduction
  • Pyrogallol / analogs & derivatives
  • Pyrogallol / chemistry
  • Pyrogallol / metabolism
  • Recombinant Proteins / biosynthesis
  • Recombinant Proteins / isolation & purification
  • Rosaniline Dyes / chemistry
  • Rosaniline Dyes / metabolism
  • Sequence Alignment
  • Solvents / chemistry
  • Substrate Specificity
  • Temperature

Substances

  • Bacterial Proteins
  • Coloring Agents
  • Recombinant Proteins
  • Rosaniline Dyes
  • Solvents
  • Pyrogallol
  • pyrogallol 1,3-dimethyl ether
  • Copper
  • ethyl violet
  • Catechol Oxidase
  • Dopamine
  • Victoria blue B

Grants and funding

This study was funded by National Nonprofit Institute Research Grant of Chinese Academy of Agricultural Sciences (No.2014-30, Funder: ZYR, http://www.iarrp.cn/sites/IARRP/), the Special Fund for Agro-scientific Research in the Public Interest (No.201403019, Funder: GQH, http://www.moa.gov.cn/), National Key Technology Research and Development Program of China (No.2013BAD05B04F02, Funder: ZYR, http://www.most.gov.cn/) and the Major State Basic Research Development Program of China (No.2012CB417104, Funder: ZYR, http://www.most.gov.cn/). ZYR and GQH designed the study and helped write the manuscript; ZYR also contributed to analyzing the data and providing reagents/materials/analysis tools.