Isolation and characterization of a heterologously expressed bacterial laccase from the anaerobe Geobacter metallireducens.
Francesca BeriniMarko VerceLuka AusecElena RosiniFabio ToninLoredano PollegioniInes Mandić-MulecPublished in: Applied microbiology and biotechnology (2018)
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.
Keyphrases
- genome wide
- escherichia coli
- bioinformatics analysis
- genome wide identification
- electron transfer
- dna methylation
- ionic liquid
- induced apoptosis
- hydrogen peroxide
- cell proliferation
- lymph node metastasis
- squamous cell carcinoma
- staphylococcus aureus
- signaling pathway
- single cell
- cystic fibrosis
- cell death
- candida albicans
- low density lipoprotein
- biofilm formation
- aqueous solution
- energy transfer