The first acidobacterial laccase-like multicopper oxidase revealed by metagenomics shows high salt and thermo-tolerance

Appl Microbiol Biotechnol. 2017 Aug;101(15):6261-6276. doi: 10.1007/s00253-017-8345-y. Epub 2017 Jun 6.

Abstract

Metagenomics is a powerful tool that allows identifying enzymes with novel properties from the unculturable component of microbiomes. However, thus far only a limited number of laccase or laccase -like enzymes identified through metagenomics has been subsequently biochemically characterized. This work describes the successful bio-mining of bacterial laccase-like enzymes in an acidic bog soil metagenome and the characterization of the first acidobacterial laccase-like multicopper oxidase (LMCO). LMCOs have hitherto been mostly studied in fungi and some have already found applications in diverse industries. However, improved LMCOs are in high demand. Using molecular screening of a small metagenomic library (13,500 clones), a gene encoding a three-domain LMCO (LacM) was detected, showing the highest similarity to putative copper oxidases of Candidatus Solibacter (Acidobacteria). The encoded protein was expressed in Escherichia coli, purified by affinity chromatography and biochemically characterized. LacM oxidized a variety of phenolic substrates, including two standard laccase substrates (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), k cat/k M = 8.45 s-1 mM-1; 2,6-dimethoxyphenol (2,6-DMP), k cat/k M = 6.42 s-1 mM-1), next to L-3,4-dihydroxyphenylalanine (L-DOPA), vanillic acid, syringaldazine, pyrogallol, and pyrocatechol. With respect to the latter two lignin building blocks, LacM showed the highest catalytic activity (k cat/k M = 173.6 s-1 mM-1) for pyrogallol, with ca. 20% activity preserved even at pH 8.0. The enzyme was thermostable and heat-activated in the interval 40-60 °C, with an optimal activity on ABTS at 50 °C. It was rather stable at high salt concentration (e.g., 34% activity preserved at 500 mM NaCl) and in the presence of organic solvents. Remarkably, LacM decolored azo and triphenylmethane dyes, also in the absence of redox mediators.

Keywords: Acidobacteria; Dye decolorization; Heterologous expression; LMCO; Laccase; Metagenomics.

MeSH terms

  • Acidobacteria / enzymology*
  • Acidobacteria / genetics*
  • Acidobacteria / metabolism
  • Bacterial Proteins / genetics
  • Cloning, Molecular
  • Escherichia coli / genetics
  • Gene Expression
  • Genomic Library
  • Hydrogen-Ion Concentration
  • Laccase / genetics*
  • Laccase / metabolism*
  • Metagenome
  • Metagenomics
  • Oxidoreductases / genetics*
  • Oxidoreductases / isolation & purification
  • Oxidoreductases / metabolism*
  • Salt Tolerance
  • Soil Microbiology*
  • Substrate Specificity
  • Temperature

Substances

  • Bacterial Proteins
  • Oxidoreductases
  • Laccase
  • copper oxidase