Skip to main content
SpringerLink
Log in

Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp

  • Biotechnologically relevant enzymes and proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

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.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Alexandre G, Zhulin IB (2000) Laccases are widespread in bacteria. Trends Biotechnol 18:41–42

    Article  CAS  PubMed  Google Scholar 

  • Ausec L, van Elsas JD, Mandic-Mulec I (2011a) Two- and three-domain bacterial laccase-like genes are present in drained peat soils. Soil Biol Biochem 43:975–983

    Article  CAS  Google Scholar 

  • Ausec L, Zakrzewski M, Goesmann A, Schlüter A, Mandic-Mulec I (2011b) Bioinformatic analysis reveals high diversity of bacterial genes for laccase-like enzymes. PLoS ONE 6(10), e25724. doi:10.1371/journal.pone.0025724

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Baldrian P (2006) Fungal laccases—occurrence and properties. FEMS Microbiol Rev 30:215–242

    Article  CAS  PubMed  Google Scholar 

  • Burgess RR (2009) Preparing a purification summary table. In: Burgess RR, Deutscher MP (eds) Methods in enzymology, vol 463. Academic Press, San Diego, pp 29–34

    Google Scholar 

  • Claus H (2003) Laccases and their occurrence in prokaryotes. Arch Microbiol 179:145–150

    CAS  PubMed  Google Scholar 

  • Durao P, Chen Z, Fernandes AT, Hildebrandt P, Murgida DH, Todorovic S, Pereira MM, Melo EP, Martins LO (2008) Copper incorporation into recombinant CotA laccase from Bacillus subtilis: characterization of fully copper loaded enzymes. J Biol Inorg Chem 13:183–193

    Article  CAS  PubMed  Google Scholar 

  • Fernandes AT, Damas JM, Todorovic S, Huber R, Baratto MC, Pogni R, Soares CM, Martins LO (2010) The multicopper oxidase from the archaeon Pyrobaculum aerophilum shows nitrous oxide reductase activity. FEBS J 277:3176–3189

    Article  CAS  PubMed  Google Scholar 

  • Foti M, Ma S, Sorokin DY, Rademaker JLW, Kuenen JG, Muyzer G (2006) Genetic diversity and biogeography of haloalkaliphilic sulphur-oxidizing bacteria belonging to the genus Thioalkalivibrio. FEMS Microbiol Ecol 56:95–101

    Article  CAS  PubMed  Google Scholar 

  • Giardina P, Faraco V, Pezzella C, Piscitelli A, Vanhulle S, Sannia G (2010) Laccases: a never-ending story. Cell Mol Life Sci 67:369–385

    Article  CAS  PubMed  Google Scholar 

  • Han MJ, Choi HT, Song HG (2005) Purification and characterization of laccase from the white rot fungus Trametes versicolor. J Microbiol 43:555–560

    CAS  PubMed  Google Scholar 

  • Hori C, Ishida T, Igarashi K, Samejima M, Suzuki H, Master E, Ferreira P, Ruiz-Dueñas FJ, Held B, Canessa P, Larrondo LF, Schmoll M, Druzhinina IS, Kubicek CP, Gaskell JA, Kersten P, St. John F, Glasner J, Sabat G, Splinter BonDurant S, Syed K, Yadav J, Mgbeahuruike AC, Kovalchuk A, Asiegbu FO, Lackner G, Hoffmeister D, Rencoret J, Gutiérrez A, Sun H, Lindquist E, Barry K, Riley R, Grigoriev IV, Henrissat B, Kües U, Berka RM, Martínez AT, Covert SF, Blanchette RA, Cullen D (2014) Analysis of the Phlebiopsis gigantea genome, transcriptome and secretome provides insight into its pioneer colonization strategies of wood. PLoS Genet 10(12):e1004759. doi:10.1371/journal.pgen.1004759

    Article  PubMed Central  PubMed  Google Scholar 

  • Käll L, Krogh A, Sonnhammer ELL (2007) Advantages of combined transmembrane topology and signal peptide prediction—the Phobius web server. Nucleic Acids Res 35:429–432

    Article  Google Scholar 

  • Kataoka K, Komori H, Ueki Y, Konno Y, Kamitaka Y, Kurose S, Tsujimura S, Higuchi Y, Kano K, Seo D, Sakurai T (2007) Structure and function of the engineered multicopper oxidase CueO from Escherichia coli—deletion of the methionine-rich helical region covering the substrate-binding site. J Mol Biol 373:141–152

    Article  CAS  PubMed  Google Scholar 

  • Kellner H, Luis P, Zimdars B, Kiesel B, Buscot F (2008) Diversity of bacterial laccase-like multicopper oxidase genes in forest and grassland Cambisol soil samples. Soil Biol Biochem 40:638–648

    Article  CAS  Google Scholar 

  • Kilaru S, Hoegger P, Kües U (2006) The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies. Curr Genet 50:45–60

    Article  CAS  PubMed  Google Scholar 

  • Lu L, Zhao M, Wang TN, Zhao LY, Du MH, Li TL, Li DB (2012) Characterization and dye decolorization ability of an alkaline resistant and organic solvents tolerant laccase from Bacillus licheniformis LS04. Bioresour Technol 115:35–40

    Article  CAS  PubMed  Google Scholar 

  • Machczynski MC, Vijgenboom E, Samyn B, Canters GW (2004) Characterization of SLAC: a small laccase from Streptomyces coelicolor with unprecedented activity. Protein Sci 13:2388–2397. doi:10.1110/ps.04759104

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277:18849–18859

    Article  CAS  PubMed  Google Scholar 

  • Miyazaki K (2005) A hyperthermophilic laccase from Thermus thermophilus HB27. Extremophiles 9:415–425

    Article  CAS  PubMed  Google Scholar 

  • Molina-Guijarro JM, Pérez J, Muñoz-Dorado J, Guillén F, Moya R, Hernández M, Arias ME (2009) Detoxification of azo dyes by a novel pH-versatile, salt-resistant laccase from Streptomyces ipomoea. Int Microbiol 12:13–21

    CAS  PubMed  Google Scholar 

  • Muyzer G, Sorokin DY, Mavromatis K, Lapidus A, Foster B, Sun H, Ivanova N, Pati A, D’haeseleer P, Woyke T, Kyrpides NC (2011) Complete genome sequence of Thioalkalivibrio sp. K90mix. Stand Genomic Sci 5:341–355

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Poklar N, Vesnaver G (2000) Thermal denaturation of proteins studied by UV spectroscopy. J Chem Educ 77:380

    Article  Google Scholar 

  • Qiagen (2003) The QIAexpressionist: a handbook for high-level expression and purification of 6xHis-tagged proteins. QIAGEN GmbH, Hilden

    Google Scholar 

  • Reiss R, Ihssen J, Thöny-Meyer L (2011) Bacillus pumilus laccase: a heat stable enzyme with a wide substrate spectrum. BMC Biotechnol 11:9. doi:10.1186/1472-6750-11-9

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Reiss R, Ihssen J, Richter M, Eichhorn E, Schilling B, Thöny-Meyer L (2013) Laccase versus laccase-like multi-copper oxidase: a comparative study of similar enzymes with diverse substrate spectra. PLoS ONE 8, e65633. doi:10.1371/journal.pone.0065633

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Ricklefs E, Winkler N, Koschorreck K, Urlacher VB (2014) Expanding the laccase-toolbox: a laccase from Corynebacterium glutamicum with phenol coupling and cuprous oxidase activity. J Biotechnol 191:46–53. doi:10.1016/j.jbiotec.2014.05.031

    Article  CAS  PubMed  Google Scholar 

  • Ruijssenaars HJ, Hartmans S (2004) A cloned Bacillus halodurans multicopper oxidase exhibiting alkaline laccase activity. Appl Microbiol Biotechnol 65:177–182

    CAS  PubMed  Google Scholar 

  • Sakasegawa S, Ishikawa H, Imamura S, Sakuraba H, Goda S, Ohshima T (2006) Bilirubin oxidase activity of Bacillus subtilis CotA. Appl Environ Microbiol 72:972–975

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Sharma P, Goel R, Capalash N (2007) Bacterial laccases. World J Microbiol Biotechnol 23:823–832

    Article  CAS  Google Scholar 

  • Shaw KL, Grimsley GR, Yakovlev GI, Makarov AA, Pace CN (2001) The effect of net charge on the solubility, activity, and stability of ribonuclease Sa. Protein Sci 10:1206–1215

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Shraddha RS, Sehgal S, Kamthania M, Kumar A (2011) Laccase: microbial sources, production, purification, and potential biotechnological applications. Enzyme Res 2011:217861. doi:10.4061/2011/217861

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Singh SK, Grass G, Rensing C, Montfort WR (2004) Cuprous oxidase activity of CueO from Escherichia coli. J Bacteriol 186:7815–7817

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Singh G, Capalash N, Goel R, Sharma P (2007) A pH-stable laccase from alkali-tolerant γ-proteobacterium JB: purification, characterization and indigo carmine degradation. Enzyme Microb Technol 41:794–799

    Article  CAS  Google Scholar 

  • Singh G, Bhalla A, Kaur P, Capalash N, Sharma P (2011) Laccase from prokaryotes: a new source for an old enzyme. Rev Environ Sci Bioechnol 10:309–326

    Article  Google Scholar 

  • Sirim D, Wagner F, Wang L, Schmid RD, Pleiss J (2011) The laccase engineering database: a classification and analysis system for laccases and related multicopper oxidases. Database 2011:bar006. doi:10.1093/database/bar006

    Article  PubMed Central  PubMed  Google Scholar 

  • Sondhi S, Sharma P, Saini S, Puri N, Gupta N (2014) Purification and characterization of an extracellular, thermo-alkali-stable, metal tolerant laccase from Bacillus tequilensis SN4. PLoS ONE 9, e96951. doi:10.1371/journal.pone.0096951

    Article  PubMed Central  PubMed  Google Scholar 

  • Sorokin DY, Kuenen JG (2005) Chemolithotrophic haloalkaliphiles from soda lakes. FEMS Microbiol Ecol 52:287–295

    Article  CAS  PubMed  Google Scholar 

  • Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol 28:2731–2739. doi:10.1093/molbev/msr121

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Theuerl S, Buscot F (2010) Laccases: toward disentangling their diversity and functions in relation to soil organic matter cycling. Biol Fertil Soils 46:215–225

    Article  CAS  Google Scholar 

  • Yang Z (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinf 9:40. doi:10.1186/1471-2105-9-40

    Article  CAS  Google Scholar 

  • Ye M, Li G, Liang W, Liu Y (2010) Molecular cloning and characterization of a novel metagenome-derived multicopper oxidase with alkaline laccase activity and highly soluble expression. Appl Microbiol Biotechnol 87:1023–1031

    Article  CAS  PubMed  Google Scholar 

  • Zheng Z, Li H, Li L, Shao W (2011) Biobleaching of wheat straw pulp with recombinant laccase from the hyperthermophilic Thermus thermophilus. Biotechnol Lett 34:541–547

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We would like to thank Dr. Zdravko Podlesek, Marko Verce, and Tjaša Prevc for help with the experiments. We are thankful to Prof. Peter Maček and Dr. Gorazd Stojkovič for constructive discussions. We would also like to thank the editor and reviewers for their suggestions that improved the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Author information

Authors and Affiliations

  1. Department of Food Science and Technology, Biotechnical Faculty, University of Ljubljana, Jamnikarjeva 101, 1000, Ljubljana, Slovenia

    Luka Ausec, Miha Črnigoj, Marko Šnajder, Nataša Poklar Ulrih & Ines Mandic-Mulec

  2. Department of Biology, Biotechnical Faculty, University of Ljubljana, Večna pot 111, 1000, Ljubljana, Slovenia

    Miha Črnigoj

  3. Centre of Excellence for Integrated Approaches in Chemistry and Biology of Proteins (CipKeBiP), Jamova 39, 1000, Ljubljana, Slovenia

    Nataša Poklar Ulrih

Authors
  1. Luka Ausec
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Miha Črnigoj
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Marko Šnajder
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nataša Poklar Ulrih
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Ines Mandic-Mulec
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Ines Mandic-Mulec.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Ausec, L., Črnigoj, M., Šnajder, M. et al. Characterization of a novel high-pH-tolerant laccase-like multicopper oxidase and its sequence diversity in Thioalkalivibrio sp. Appl Microbiol Biotechnol 99, 9987–9999 (2015). https://doi.org/10.1007/s00253-015-6843-3

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-015-6843-3

Keywords

Search

Navigation