In the transition to the post-petroleum economy, there is a growing demand for novel enzymes with high process performances to replace traditional chemistry with a more 'green' approach. To date, microorganisms encompass the richest source of industrial biocatalysts, but the Earth-living microbiota remains largely untapped by using traditional isolation and cultivation methods. Metagenomics, which is culture independent, represents a powerful tool for discovering novel enzymes from unculturable microorganisms. Herein, we summarize the variety of approaches adopted for mining environmental DNA and, based on a systematic literature review, we provide a comprehensive list of 332 industrially relevant enzymes discovered from metagenomes within the last three years.

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 /k = 8.45 s mM; 2,6-dimethoxyphenol (2,6-DMP), k /k = 6.42 s mM), 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 /k = 173.6 s mM) 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.

Graphical abstractTreatment of 50 μM RBBR with 60 μg of N. gerenzanensis peroxidase preparation: the dye decolorizing activity was clearly observed.