Skip to main content
SpringerLink
Log in

Thermostable and highly specific l-aspartate oxidase from Thermococcus litoralis DSM 5473: cloning, overexpression, and enzymological properties

  • Original Paper
  • Published:
Extremophiles Aims and scope Submit manuscript

Abstract

We successfully expressed the l-aspartate oxidase homolog gene (accession no: OCC_06611) of Thermococcus litoralis DSM 5473 in the soluble fraction of Escherichia coli BL21 (DE3) using a pET21b vector with 6X His tag at its C-terminus. The gene product (Tl-LASPO) showed l-aspartate oxidase activity in the presence of FAD in vitro, and this report is the first that details an l-aspartate oxidase derived from a Thermococcus species. The homologs of Tl-LASPO existed mainly in archaea, especially in the genus of Thermococcus, Pyrococcus, Sulfolobus, and Halobacteria. The quaternary structure of Tl-LASPO was homotrimeric with a subunit molecular mass of 52 kDa. The enzyme activity of Tl-LASPO increased with temperature up to 70 °C. Tl-LASPO was active from pH 6.0 to 9.0, and its highest activity was at pH 8.0. Tl-LASPO was stable at 80 °C for 1 h. The highest k cat/K m value was observed in assays at 70 °C. Tl-LASPO was highly specific for l-aspartic acid. Tl-LASPO utilized fumaric acid, 2,6-dichlorophenolindophenol, and ferricyanide in addition to FAD as a cofactor under anaerobic conditions. The absorption spectrum of holo-Tl-LASPO exhibited maxima at 380 and 450 nm. The FAD dissociation constant, K d, of the FAD-Tl-LASPO complex was determined to be 5.9 × 10−9 M.

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
Fig. 6
Fig. 7

Similar content being viewed by others

Abbreviations

DCIP:

2,6-Dichlorophenolindophenol

DTT:

Dithiothreitol

DTNB:

Dithiobis-(2-nitrobenzoic acid)

EDTA:

Ethylenediaminetetraacetic acid

FAD:

Flavin adenine dinucleotide

KPB:

Potassium phosphate buffer

LB:

Luria–Bertani

MALDI-TOF-MS:

Matrix-assisted laser desorption–ionization mass spectrometry

NAD:

Nicotine adenine dinucleotide

PLP:

Pyridoxal 5′-phosphate

PCR:

Polymerase chain reaction

References

  • Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    Article  CAS  PubMed  Google Scholar 

  • Arroyo M, Menendez M, Garcia JL, Campillo N, Hormigo D, de Mata I I, Castillon MP, Acebal C (2007) The role of cofactor binding in tryptophan accessibility and conformational stability of His-tagged d-amino acid oxidase from Trigonopsis variabilis. Biochim Biophys Acta 1774:556–565

    Article  CAS  PubMed  Google Scholar 

  • Bifulco D, Pollegioni L, Tessaro D, Servi S, Molla G (2013) A thermostable l-aspartate oxidase: a new tool for biotechnological applications. Appl Microbiol Biotechnol 97:7285–7295

    Article  CAS  PubMed  Google Scholar 

  • Bohmer A, Muller A, Passarge M, Liebs P, Honeck H, Muller HG (1989) A novel l-glutamate oxidase from Streptomyces endus. Purification and properties. Eur J Biochem 182:327–332

    Article  CAS  PubMed  Google Scholar 

  • Braga MD, Martins AM, Amora DN, de Menezes DB, Toyama MH, Toyama DO, Marangoni S, Alves CD, Barbosa PS, de Sousa Alves R, Fonteles MC, Monteiro HS (2008) Purification and biological effects of l-amino acid oxidase isolated from Bothrops insularis venom. Toxicon 51:199–207

    Article  CAS  PubMed  Google Scholar 

  • Flachmann R, Kunz N, Seifert J, Gutlich M, Wientjes FJ, Laufer A, Gassen HG (1988) Molecular biology of pyridine nucleotide biosynthesis in Escherichia coli. Cloning and characterization of quinolinate synthesis genes nadA and nadB. Eur J Biochem 175:221–228

    Article  CAS  PubMed  Google Scholar 

  • Geueke B, Weckbecker A, Hummel W (2007) Overproduction and characterization of a recombinant d-amino acid oxidase from Arthrobacter protophormiae. Appl Microbiol Biotechnol 74:1240–1247

    Article  CAS  PubMed  Google Scholar 

  • Hahn K, Neumeister K, Mix A, Kottke T, Groger H, Fischer von Mollard G (2017) Recombinant expression and characterization of a l-amino acid oxidase from the fungus Rhizoctonia solani. Appl Microbiol Biotechnol 101:2853–2864

    Article  CAS  PubMed  Google Scholar 

  • Isobe K, Sugawara A, Domon H, Fukuta Y, Asano Y (2012) Purification and characterization of an l-amino acid oxidase from Pseudomonas sp. AIU 813. J Biosci Bioeng 114:257–261

    Article  CAS  PubMed  Google Scholar 

  • Katoh A, Uenohara K, Akita M, Hashimoto T (2006) Early steps in the biosynthesis of NAD in Arabidopsis start with aspartate and occur in the plastid. Plant Physiol 141:851–857

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Leese C, Fotheringham I, Escalettes F, Speight R, Grogan G (2013) Cloning, expression, characterisation and mutational analysis of l-aspartate oxidase from Pseudomonas putida. J Mol Catal B 85–86:17–22

    Article  Google Scholar 

  • Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56:658–666

    Article  CAS  Google Scholar 

  • Marinoni I, Nonnis S, Monteferrante C, Heathcote P, Hartig E, Bottger LH, Trautwein AX, Negri A, Albertini AM, Tedeschi G (2008) Characterization of l-aspartate oxidase and quinolinate synthase from Bacillus subtilis. FEBS J 275:5090–5107

    Article  CAS  PubMed  Google Scholar 

  • Mot YY, Othman I, Sharifah SH (2017) Synergistic antibacterial effect of co-administering adipose-derived mesenchymal stromal cells and Ophiophagus hannah l-amino acid oxidase in a mouse model of methicillin-resistant Staphylococcus aureus-infected wounds. Stem Cell Res Ther 8:5

    Article  PubMed  PubMed Central  Google Scholar 

  • Neuner A, Jannasch HW, Belkin S, Stetter KO (1990) Thermococcus litoralis sp. nov.: a new species of extremely thermophilic marine archaebacterial. Arch Microbiol 153:205–207

    Article  Google Scholar 

  • Nishikawa T (2005) Metabolism and functional roles of endogenous d-serine in mammalian brains. Biol Pharm Bull 28:1561–1565

    Article  CAS  PubMed  Google Scholar 

  • Nishioka M, Mizuguchi H, Fujiwara S, Komatsubara S, Kitabayashi M, Uemura H, Takagi M, Imanaka T (2001) Long and accurate PCR with a mixture of KOD DNA polymerase and its exonuclease deficient mutant enzyme. J Biotechnol 88:141–149

    Article  CAS  PubMed  Google Scholar 

  • Patsoukis N, Georgiou CD (2004) Determination of the thiol redox state of organisms: new oxidative stress indicators. Anal Bioanal Chem 378:1783–1792

    Article  CAS  PubMed  Google Scholar 

  • Pilone SM, Pollegioni L, Casalin P, Curti B, Ronchi S (1989) Properties of d-amino-acid oxidase from Rhodotorula gracilis. Eur J Biochem 180:199–204

    Article  Google Scholar 

  • Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4:406–425

    CAS  PubMed  Google Scholar 

  • Sakuraba H, Satomura T, Kawakami R, Yamamoto S, Kawarabayasi Y, Kikuchi H, Ohshima T (2002) l-Aspartate oxidase is present in the anaerobic hyperthermophilic archaeon Pyrococcus horikoshii OT-3: characteristics and role in the de novo biosynthesis of nicotinamide adenine dinucleotide proposed by genome sequencing. Extremophiles 6:275–281

    Article  CAS  PubMed  Google Scholar 

  • Sakuraba H, Yoneda K, Asai I, Tsuge H, Katunuma N, Ohshima T (2008) Structure of l-aspartate oxidase from the hyperthermophilic archaeon Sulfolobus tokodaii. Biochim Biophys Acta 1784:563–571

    Article  CAS  PubMed  Google Scholar 

  • Schell MJ, Cooper OB, Snyder SH (1997) d-Aspartate localizations imply neuronal and neuroendocrine roles. Proc Natl Acad Sci USA 94:2013–2018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Seifert J, Kunz N, Flachmann R, Laufer A, Jany KD, Gassen HG (1990) Expression of the E. coli nadB gene and characterization of the gene product l-aspartate oxidase. Biol Chem Hoppe Seyler 371:239–248

    Article  CAS  PubMed  Google Scholar 

  • Stabeli RG, Marcussi S, Carlos GB, Pietro RC, Selistre-de-Araujo HS, Giglio JR, Oliveira EB, Soares AM (2004) Platelet aggregation and antibacterial effects of an l-amino acid oxidase purified from Bothrops alternatus snake venom. Bioorg Med Chem 12:2881–2886

    Article  CAS  PubMed  Google Scholar 

  • Tachibana Y, Takaha T, Fujiwara S, Takagi M, Imanaka T (2000) Acceptor specificity of 4-alpha-glucanotransferase from Pyrococcus kodakaraensis KOD1, and synthesis of cycloamylose. J Biosci Bioeng 90:406–409

    Article  CAS  PubMed  Google Scholar 

  • Takahashi S, Furukawara M, Omae K, Tadokoro N, Saito Y, Abe K, Kera Y (2014) A highly stable d-amino acid oxidase of the thermophilic bacterium Rubrobacter xylanophilus. Appl Environ Microbiol 80:7219–7229

    Article  PubMed  PubMed Central  Google Scholar 

  • Thompson JD, Gibson TJ, Higgins DG (2002) Multiple sequence alignment using ClustalW and ClustalX. Curr Protoc Bioinform Chapter 2, Unit 2.3

  • Vargas LJ, Quintana JC, Pereanez JA, Nunez V, Sanz L, Calvete J (2013) Cloning and characterization of an antibacterial l-amino acid oxidase from Crotalus durissus cumanensis venom. Toxicon 64:1–11

    Article  CAS  PubMed  Google Scholar 

  • Washio T, Kato S, Oikawa T (2016) Molecular cloning and enzymological characterization of pyridoxal 5′-phosphate independent aspartate racemase from hyperthermophilic archaeon Thermococcus litoralis DSM 5473. Extremophiles 20(5):711–721

    Article  CAS  PubMed  Google Scholar 

  • Wellner D, Meister A (1960) Crystalline l-amino acid oxidase of Crotalus adamanteus. J Biol Chem 235:2013–2018

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study was supported in part by the Ministry of Education, Culture, Sports, Science and Technology (MEXT)—Supported Program for the Strategic Research Foundation at Private Universities (Project no. S1311044), 2013–2017.

Author information

Authors and Affiliations

  1. Department of Life Science and Biotechnology, Faculty of Chemistry, Materials and Bioengineering, Kansai University, 3-3-35 Yamate-Cho, Suita, Osaka, 564-8680, Japan

    Tsubasa Washio & Tadao Oikawa

  2. Kansai University High Technology Research Center, 3-3-35 Yamate-Cho, Suita, Osaka, 564-8680, Japan

    Tadao Oikawa

Authors
  1. Tsubasa Washio
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tadao Oikawa
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Tadao Oikawa.

Additional information

Communicated by H. Atomi.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Washio, T., Oikawa, T. Thermostable and highly specific l-aspartate oxidase from Thermococcus litoralis DSM 5473: cloning, overexpression, and enzymological properties. Extremophiles 22, 59–71 (2018). https://doi.org/10.1007/s00792-017-0977-4

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00792-017-0977-4

Keywords

Search

Navigation