Skip to main content
SpringerLink
Log in

Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A constitutively expressed thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila was purified to homogeneity by applying hydrophobic interaction, anion exchange and gel filtration chromatography, giving a yield of 26% and a specific activity of 19.5 units mg−1. The purified enzyme has an estimated molecular mass of 108 kDa and an isoelectric point of 4.2. The amidase is active at a broad pH range (pH 4–9) and temperature range (40–80°C) and has a half-life of 1.2 h at 70°C. Inhibition of enzyme activity was observed in the presence of metal ions, such as Co2+, Hg2+, Cu2+, Ni2+, and thiol reagents. The amidase has a broad substrate spectrum, including aliphatic, aromatic and amino acid amides. The presence of a double bond or a methyl group near the carboxamide group of aliphatic and amino acid amides enhances the enzymatic activity. Among aromatic amides with substitutions at the o-, m-, or p-position, the p-substituted amides are the preferred substrates. The highest acyl transferase activity was detected with hexanoamide, isobutyramide and propionamide. The K m values for propionamide, methacrylamide, benzamide and 2-phenylpropionamide are 7.4, 9.2, 4.9 and 0.9 mM, respectively. The amidase is highly S-stereoselective for 2-phenylpropionamide; and the racemic amide was converted to the corresponding S-acid with an enantiomeric excess of >95% at 50% conversion of the substrate. In contrast, the d,l-tryptophanamide and d,l-methioninamide were converted to the corresponding d,l-acids at the same rate. This thermostable enzyme represents the first reported amidase from a thermophilic actinomycete.

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

Similar content being viewed by others

References

  • d’Abusco AS, Ammendola S, Scandurra R, Politi L (2001) Molecular and biochemical characterization of the recombinant amidase from hyperthermophilic archaeon Sulfolobus solfataricus. Extremophiles 5:183–192

    CAS  PubMed  Google Scholar 

  • Alcantara A-R, Sanchez-Montero JM, Sinisterra JV (2000) Chemoenzymatic preparation of enantiomerically pure S(+)-2-arylpropionic acids with anti-inflammatory activity. In: Patel RN (ed) Stereoselective biocatalysis. Dekker, New York, pp 659–702

  • Arcus C, Kenyon J (1939) The mechanism of the Hofman reaction. Retention of optical activity during the reaction with (+)-hydratopamide. J Chem Soc 916–920

  • Baek DH, Song JJ, Lee SG, Kwon SJ, Asano Y, Sung MH (2003) New thermostable d-methionine amidase from Brevibacillus borstelensis BCS-1 and its application for d-phenylalanine production. Enzyme Microb Technol 32:131–139

    Article  CAS  Google Scholar 

  • Bhalla T, Kumar J, Kumar H, Agrawal HO (1997) Amidase production by Rhodococcus sp. NHB-2. Sci Lett 11–12:139–142

    Google Scholar 

  • Boshoff H, Mizrahi V (1998) Purification, gene cloning, targeted knockout, overexpression, and biochemical characterization of the major pyrazinamidase from Mycobacterium smegmatis. J Bacteriol 180:5809–5814

    CAS  PubMed  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  • Chebrou H, Bigey F, Arnaud A, Galzy P (1996) Study of the amidase signature group. Biochim Biophys Acta 1298:285–293

    CAS  PubMed  Google Scholar 

  • Cheong T, Oriel PJ (2000) Cloning of a wide-spectrum amidase from Bacillus stearothermophilus BR388 in Escherichia coli and marked enhancement of amidase expression using directed evolution. Enzyme Microb Technol 26:152–158

    CAS  PubMed  Google Scholar 

  • Ciskanik L, Wilczek JM, Fallon RD (1995) Purification and characterization of an enantioselective amidase from Pseudomonas chlororaphis B23. Appl Environ Microbiol 61:998–1003

    CAS  Google Scholar 

  • Egorova KV, Trauthwein H, Antranikian G (2002) A rapid and sensitive method for the detection of amidases in polyacrylamide gels and agar plates. German patent 10233686.5

  • Fournand D, Arnaud A, Galzy P (1998a) Study of the acyl transfer activity of a recombinant amidase overproduced in an Escherichia coli strain. Application for short-chain hydroxamic acid and acid hydrazide synthesis. J Mol Catal 4:77–90

    Article  CAS  Google Scholar 

  • Fournand D, Bigey F, Arnaud A (1998b) Acyl transfer activity of an amidase from Rhodococcus sp. strain R312: formation of a wide range of hydroxamic acids. Appl Environ Microbiol 64:2844–2852

    CAS  PubMed  Google Scholar 

  • Fraser JA, Davis MA, Hynes MJ (2002) The genes gmdA, encoding an amidase, and bzuA, encoding a cytochrome P450, are required for benzamide utilization in Aspergillus nidulans. Exp Mycol 2:135–146

    Google Scholar 

  • Gilligan T, Yamada H, Nagasawa T (1993) Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328. Appl Microbiotechnol 6:720–725

    Google Scholar 

  • Hermes H, Tandler RF, Sonke T, Dijkhuizen L, Meijer EM (1994) Purification and characterization of an l-amino amidase from Mycobacterium neoaurum ATCC 25795. Appl Environ Microbiol 1:153–159

    Google Scholar 

  • Hirrlinger B, Stolz A (1997) Formation of a chiral hydroxamic acid with an amidase from Rhodococcus erythropolis MP50 and subsequent chemical rearrangement to a chiral amine. Appl Environ Microbiol 9:3390–3393

    Google Scholar 

  • Hirrlinger B, Stolz A, Knackmuss HJ (1996) Purification and properties of an amidase from Rhodococcus erythropolis MP50 which enantioselectively hydrolyzes 2-arylpropionamides. J Bacteriol 178:3501–3507

    CAS  PubMed  Google Scholar 

  • Joeres U, Kula MR (1994) Purification and characterisation of a microbial l-carnitine amidase. Appl Microbiol Biotechnol 40:606–610

    Article  CAS  PubMed  Google Scholar 

  • Kakeya H, Sakai N, Sugai T, Ohta H (1991) Microbial hydrolysis as a potent method for the preparation of optically active nitriles, amides and carboxylic acids. Tetrahedron Lett 32:1343–1346

    Article  CAS  Google Scholar 

  • Kamphuis J, Boesten WHJ, Kaptein B, Hermes HFM, Sonke T, Broxterman QB, Tweel WJJ van den, Schoemaker HE (1992) The production and uses of optically pure natural and unnatural amino acids. In: Collins AN, Sheldrake GN, Crosby J (eds) Chirality in industry: the commercial manufacture and applications of optically active compounds. Dekker, New York, pp 187–208

  • Kobayashi M, Komeda H, Nagasawa T, Nishiyama M, Horinouchi S, Beppu T, Yamada H, Shimizu S (1993) Amidase coupled with low-molecular-mass nitrile hydratase from Rhodococcus rhodochrous J1. Sequencing and expression of the gene and purification and characterization of the gene product. Eur J Biochem 217:327–336

    CAS  PubMed  Google Scholar 

  • Komeda H, Asano Y (2000) Gene cloning, nucleotide sequencing, and purification and characterization of the d-stereospecific amino-acid amidase from Ochrobactrum anthropi SV3. Eur J Biochem 267:2028–2035

    CAS  PubMed  Google Scholar 

  • Kotlova E, Chestukhina GG, Astaurova OB, Leonova TE, Yanenko AS, Debabov VG (1999) Isolation and primary characterization of an amidase from Rhodococcus rhodochrous. Biochemistry (Mosc) 64:384–389

    Google Scholar 

  • Krieg L, Ansorge-Schumacher MB, Kula MR (2002) Screening for amidases: isolation and characterisation of a novel d-amidase from Variovorax paradoxus. Adv Synth Catal 344:965–973

    Article  CAS  Google Scholar 

  • Labahn J, Neumann S, Buldt G, Kula MR, Granzin J (2002) An alternative mechanism for amidase signature enzymes. J Mol Biol 322:1053–1064

    Article  CAS  PubMed  Google Scholar 

  • Laemmli U (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685

    PubMed  Google Scholar 

  • Layh N, Hirrlinger B, Stolz A, Knackmuss HJ (1997) Enrichment strategies for nitrile-hydrolysing bacteria. Appl Microbiol Biotechnol 47:668–674

    Article  CAS  Google Scholar 

  • Mayaux J, Cerebelaud E, Soubrier F, Faucher D, Petre D (1990) Purification, cloning, and primary structure of an enantiomer-selective amidase from Brevibacterium sp. strain R312: structural evidence for genetic coupling with nitrile hydratase. J Bacteriol 172:6764–6773

    CAS  PubMed  Google Scholar 

  • Mayaux J, Cerbelaud E, Soubrier F, Yeh P, Blanche F, Petre D (1991) Purification, cloning, and primary structure of a new enantiomer-selective amidase from a Rhodococcus strain: structural evidence for a conserved genetic coupling with nitrile hydratase. J Bacteriol 173:6694–6704

    CAS  PubMed  Google Scholar 

  • Nawaz M, Davis JW, Wolfram JH, Chapatwala KD (1991) Degradation of organic cyanides by Pseudomonas aeruginosa. Appl Biochem Biotechnol 28/29:865–875

    Google Scholar 

  • Nawaz M, Khan AA, Seng JE, Leakey JE, Siitonen PH, Cerniglia CE (1994) Purification and characterization of an amidase from an acrylamide-degrading Rhodococcus sp. Appl Environ Microbiol 60:3343–3348

    CAS  PubMed  Google Scholar 

  • Nawaz M, Khan AA, Bhattacharayya D, Siitonen PH, Cerniglia CE (1996) Physical, biochemical, and immunological characterization of a thermostable amidase from Klebsiella pneumoniae NCTR 1. J Bacteriol 178:2397–2401

    CAS  PubMed  Google Scholar 

  • Neumann S, Kula MR (2002) Gene cloning, overexpression and biochemical characterization of the peptide amidase from Stenotrophomonas maltophilia. Appl Microbiol Biotechnol 58:773–780

    Google Scholar 

  • Ozaki A, Kawasaki H, Yagasaki M, Hashimoto Y (1992) Enzymatic production of d-alanine from d,l-alaninamide by novel d-alaninamide specific amide hydrolase. Biosci Biotechnol Biochem 56:1980–1984

    CAS  Google Scholar 

  • Silman N, Carver MA, Jones CW (1991) Directed evolution of amidase in Methylophilus methylotrophus: purification and properties of amidases from wild-type and mutant strain. J Gen Microbiol 13:169–178

    Google Scholar 

  • Skouloubris S, Labigne A, De Reuse H (1997) Identification and characterization of an aliphatic amidase in Helicobacter pylori. Mol Microbiol 25:989–998

    CAS  PubMed  Google Scholar 

  • Sonke T, Kaptein B, Boesten WHJ, Boxterman QB, Schoemaker HE, Kamphuis J, Formaggio F, Toniolo C, Rutjes FP (2000) Aminoamidase-catalyzed preparation and further transformations of enantiopure a-hydrogen and a,a-disubstituted a-amino acids. In: Ronda P (ed) Stereoselective biocatalysis. Dekker, New York, pp 23–58

  • Trott S, Bauer R, Knackmuss HJ, Stolz A (2001) Genetic and biochemical characterization of an enantioselective amidase from Agrobacterium tumefaciens strain d3. Microbiology 147:1815–1824

    CAS  PubMed  Google Scholar 

  • Webster N, Ramsden DK, Hughes J (2001) Comparative characterization of two Rhodococcus species as potential biocatalysts for ammonium acrylate production. Biotechnol Lett 23:95–101

    Article  CAS  Google Scholar 

  • Yamaki T, Oikawa T, Ito K, Nakamura T (1997) Cloning and sequencing of a nitrile hydratase gene from Pseudonocardia thermophila JCM3095. J Ferment Bioeng 83:474–477

    CAS  Google Scholar 

  • Yamamoto K, Otsubo K, Matsuo A, Hayashi T, Fujimatsu I, Komatsu KI (1996) Production of R-(−)-ketoprofen from an amide compound by Comamonas acidovorans KPO-2771-4. Appl Environ Microbiol 1:152–155

    Google Scholar 

Download references

Acknowledgements

We gratefully acknowledge the technical help of Mrs. N. Rudolph and the financial support of the Fonds der Chemischen Industrie.

Author information

Authors and Affiliations

  1. Institute of Technical Microbiology, Technical University Hamburg–Harburg, Kasernenstr. 12, 21073, Hamburg, Germany

    Ksenia Egorova & Garabed Antranikian

  2. Project House Catalysis, DEGUSSA AG, 65926, Frankfurt am Main, Germany

    Harald Trauthwein

  3. Project House Biotechnology, DEGUSSA AG, 63457, Hanau-Wolfgang, Germany

    Stefan Verseck

Authors
  1. Ksenia Egorova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Harald Trauthwein
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Stefan Verseck
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Garabed Antranikian
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Garabed Antranikian.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Egorova, K., Trauthwein, H., Verseck, S. et al. Purification and properties of an enantioselective and thermoactive amidase from the thermophilic actinomycete Pseudonocardia thermophila . Appl Microbiol Biotechnol 65, 38–45 (2004). https://doi.org/10.1007/s00253-004-1607-5

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-004-1607-5

Keywords

Search

Navigation