Applied Microbiology and Biotechnology

, Volume 102, Issue 5, pp 2425–2439 | Cite as

Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens

  • Francesca Berini
  • Marko Verce
  • Luka Ausec
  • Elena Rosini
  • Fabio Tonin
  • Loredano Pollegioni
  • Ines Mandić-Mulec
Environmental biotechnology


Bioinformatics has revealed the presence of putative laccase genes in diverse bacteria, including extremophiles, autotrophs, and, interestingly, anaerobes. Integrity of laccase genes in anaerobes has been questioned, since laccases oxidize a variety of compounds using molecular oxygen as the electron acceptor. The genome of the anaerobe Geobacter metallireducens GS-15 contains five genes for laccase-like multicopper oxidases. In order to show whether one of the predicted genes encodes a functional laccase, the protein encoded by GMET_RS10855 was heterologously expressed in Escherichia coli cells. The His6-tagged enzyme (named GeoLacc) was purified to a large extent in the apoprotein, inactive form: incubation with CuSO4 allowed a 43-fold increase of the specific activity yielding a metallo-enzyme. The purified enzyme oxidized some of the typical laccase substrates, including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), syringaldazine, and 2,6-dimethoxyphenol (2,6-DMP), along with pyrogallol and K4[Fe(CN)6]. Temperature optimum was 75 °C and pH optimum for ABTS and 2,6-DMP oxidation was ~ 6.0. As observed for other laccases, the enzyme was inhibited by halide anions and was sensitive to increasing concentrations of dimethyl sulfoxide and Tween-80. Notably, GeoLacc possesses a very high affinity for dioxygen: a similar activity was measured performing the reaction at air-saturated or microaerophilic conditions.


Bacterial laccase Characterization Bioinformatics Heterologous expression Anaerobic microorganisms 



The authors thank Derek R. Lovley of the University of Massachusetts for providing the genomic DNA of Geobacter metallireducens GS-15.


This research was financially supported by the research grants P4-0116 and J4-4250 awarded to IMM by Slovenian Research Agency.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of Biotechnology and Life SciencesUniversity of InsubriaVareseItaly
  2. 2.‘The Protein Factory Research Center’Politecnico di Milano and University of InsubriaMilanItaly
  3. 3.Department of Food Science and Technology, Biotechnical FacultyUniversity of LjubljanaLjubljanaSlovenia
  4. 4.Department of Bio-engineering SciencesVrije Universiteit BrusselBrusselBelgium
  5. 5.Genialis d.o.oLjubljanaSlovenia
  6. 6.Department of BiotechnologyDelft University of TechnologyDelftThe Netherlands

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