title: Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp.
abstract: Laccases are oxidoreductases mostly studied in fungi, while bacterial laccases remain poorly studied despite their high genetic diversity and potential for biotechnological application. Our previous bioinformatic analysis identified alkaliphilic bacterial strains Thioalkalivibrio sp. as potential sources of robust bacterial laccases that would be stable at high pH. In the present work, a gene for a laccase-like enzyme from Thioalkalivibrio sp. ALRh was cloned and expressed as a 6× His-tagged protein in Escherichia coli. The purified enzyme was a pH-tolerant laccase stable in the pH range between 2.1 and 9.9 at 20 °C as shown by intrinsic fluorescence emission spectrometry. It had optimal activities at pH 5.0 and pH 9.5 with the laccase substrates 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and 2,6-dimethoxyphenol, respectively. In addition, it could oxidize several other monophenolic compounds and potassium hexacyanoferrate(II) but not tyrosine. It showed highest activity at 50 °C, making it suitable for prolonged incubations at this temperature. The present study shows that Thioalkalivibrio sp. encodes an active, alkaliphilic, and thermo-tolerant laccase and contributes to our understanding of the versatility of bacterial laccase-like multicopper oxidases in general.
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.
authors.forename: Luka
authors.initials: L
authors.lastname: Ausec
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.|Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Miha
authors.initials: M
authors.lastname: Črnigoj
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.
authors.forename: Marko
authors.initials: M
authors.lastname: Šnajder
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia.|Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, 1000, Ljubljana, Slovenia.
authors.forename: Nataša Poklar
authors.initials: NP
authors.lastname: Ulrih
authors.affiliation: Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia. Ines.MandicMulec@bf.uni-lj.si.
authors.forename: Ines
authors.initials: I
authors.lastname: Mandic-Mulec
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D016000:Cluster Analysis; D042001:Ectothiorhodospiraceae / Q000201:enzymology* / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D014644:Genetic Variation; D006863:Hydrogen-Ion Concentration; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011993:Recombinant Fusion Proteins; D042845:Laccase
keywords: Bacterial laccases; Enzyme characterization; Laccase-like multicopper oxidases (LMCO); Laccases; Thioalkalivibrio