Enzymatic Detection of d-Amino Acids

  • Gianluca MollaEmail author
  • Luciano Piubelli
  • Federica Volontè
  • Mirella S. Pilone
Part of the Methods in Molecular Biology book series (MIMB, volume 794)


d-Amino acids play several key roles and are widely diffused in living organisms, from bacteria (in which d-alanine is a component of the cell wall) to mammals (where d-serine is involved in glutamatergic neurotransmission in the central nervous system). The study of the biological processes involving d-amino acids and their use as clinical or biotechnological biomarkers requires reliable methods of quantifying them. Although “traditional” analytical techniques have been (and still are) employed for such tasks, enzymatic assays based on enzymes which possess a strict stereospecificity (i.e., that are only active on the d-enantiomers of amino acids) allowed the set-up of low-cost protocols with a high sensitivity and selectivity and suitable for determining the d-amino acid content of complex biological samples. The most exploited enzyme in these assays is d-amino acid oxidase, a flavoenzyme that exclusively oxidizes d-amino acids and possesses with a broad substrate specificity and a high kinetic efficiency.

Key words

Enzymatic assay d-Amino acid oxidase Coupled assay Spectrophotometry Analytical detection 



This work was supported by grants from Fondo di Ateneo per la Ricerca (University of Insubria) to G. Molla and L. Piubelli.


  1. 1.
    Auclair J L, Patton R L (1950) On the occurrence of D-alanine in the haemolymph of the milkweed bug Oncopeltus fasciatus. Rev Can Biol 9, 3–8.PubMedGoogle Scholar
  2. 2.
    Corrigan J J (1969) D-amino acids in animals. Science 164, 142–149.PubMedCrossRefGoogle Scholar
  3. 3.
    Helfman PM, Bada J L, Shou M Y (1977) Considerations on the role of aspartic acid racemization in the aging process. Gerontology 23, 419–425.PubMedCrossRefGoogle Scholar
  4. 4.
    Hashimoto A, Nishikawa T, Oka T et al. (1992) Determination of free amino acid enantiomers in rat brain and serum by high-performance liquid chromatography after derivatization with N-tert-butyloxycarbonyl-L-cysteine and o-phthaldialdehyde. J Chromatogr 582, 41–48.PubMedCrossRefGoogle Scholar
  5. 5.
    Snyder S H, Kim P M (2000) D-amino acids as putative neurotransmitters: focus on D-serine. Neurochem Res 25, 553–560.PubMedCrossRefGoogle Scholar
  6. 6.
    Oliet S H, Mothet J P (2006) Molecular determinants of D-serine-mediated gliotransmission: from release to function. Glia 54, 726–737.PubMedCrossRefGoogle Scholar
  7. 7.
    Pollegioni L, Sacchi S (2010) Metabolism of the neuromodulator D-serine. Cell Mol Life Sci 67, 2387–2404.PubMedCrossRefGoogle Scholar
  8. 8.
    Rubio-Barroso S, Santos-Delgado M J, Martin-Olivar C, Polo-Diez L M (2006) Indirect chiral HPLC determination and fluorimetric detection of D-amino acids in milk and oyster samples. J Dairy Sci 89, 82–89.PubMedCrossRefGoogle Scholar
  9. 9.
    Friedman M J (1999) Chemistry, nutrition, and microbiology of D-amino acids. Agric Food Chem 47, 3457–3479.CrossRefGoogle Scholar
  10. 10.
    Marchelli R, Palla G, Dossena A et al. (1997) D-ammino acidi: marker molecolari di stagionatura e di tipicità per il Parmigiano-Reggiano e il Grana Padano. Scienza Tecnica Lattiero-Casearia 48, 21–32.Google Scholar
  11. 11.
    Caligiuri, A., D’Arrigo, P., Rosini, E. et al. (2006) Enzymatic conversion of unnatural amino acids by yeast D-amino acid oxidase. Adv Synth Catal 348, 2183–2190.CrossRefGoogle Scholar
  12. 12.
    Pollegioni L., Piubelli L., Sacchi S et al. (2007) Physiological functions of D-amino acid oxidases: from yeast to humans. Cell Mol Life Sci 64, 1373–1394.PubMedCrossRefGoogle Scholar
  13. 13.
    Sacchi S, Lorenzi S, Molla G et al. (2002) Engineering the substrate specificity of D-amino-acid oxidase. J Biol Chem 277, 27510–27516.PubMedCrossRefGoogle Scholar
  14. 14.
    Sacchi S, Rosini E, Molla G et al. (2004) Modulating D-amino acid oxidase substrate specificity: production of an enzyme for analytical determination of all D-amino acids by directed evolution. Protein Eng Des Sel 17, 517–25.PubMedCrossRefGoogle Scholar
  15. 15.
    Oguri S, Nomura M, Fujita Y (2005) A new strategy for the selective determination of D-amino acids: enzymatic and chemical modifications for pre-column derivatization. J Chromatogr A 1078, 51–58.PubMedCrossRefGoogle Scholar
  16. 16.
    Rosini E, Molla G, Rossetti C et al. (2008) A biosensor for all D-amino acids using evolved D-amino acid oxidase. J Biotechnol 135, 377–384.PubMedCrossRefGoogle Scholar
  17. 17.
    Pernot P, Mothet J P, Schuvailo O et al. (2008) Characterization of a yeast D-amino acid oxidase microbiosensor for D-serine detection in the central nervous system. Anal Chem 80, 1589–1597.PubMedCrossRefGoogle Scholar
  18. 18.
    Molla G, Vegezzi C, Pilone MS, Pollegioni L (1998) Overexpression in Escherichia coli of a recombinant chimeric Rhodotorula gracilis D-amino acid oxidase. Protein Expr Purif 14, 289–294.PubMedCrossRefGoogle Scholar
  19. 19.
    Fantinato S, Pollegioni L, Pilone M S (2001) Engineering, expression and purification of a His-tagged chimeric D-amino acid oxidase from Rhodotorula gracilis. Enzyme Microb Technol 29, 407–412.CrossRefGoogle Scholar
  20. 20.
    Negri A, Massey V, Williams C H Jr (1987) D-Aspartate oxidase from beef kidney: purification and properties. J Biol Chem 262, 10026–10034.PubMedGoogle Scholar
  21. 21.
    Simonic T, Duga S, Negri A et al. (1997) cDNA cloning and expression of the flavoprotein D-aspartate oxidase from bovine kidney cortex. Bioch J 322, 729–735.Google Scholar
  22. 22.
    Bohme A, Winkler O (1954) Zur Bestimmung geringer Mengen Acetaldehyd. Z Anal Chem 412, 1–5.CrossRefGoogle Scholar
  23. 23.
    Walsh C T, Schonbrunn A, Abeles R H (1971) Studies on the mechanism of action of D-amino acid oxidase: evidence for removal of substrate α-hydrogen as a proton. J Biol Chem 216, 6855–6866.Google Scholar
  24. 24.
    Chlumsky L J, Zhang L, Rhamsey A J, Schuman Jorns M (1993) Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine. Biochemistry 32, 11132–11142.PubMedCrossRefGoogle Scholar
  25. 25.
    Mori N, Sano M, Tani Y, Yamada H (1980) Purification and properties of sarcosine oxidase from Cylindrocarpon didymum M-1. Agric Biol Chem 44, 1391–1397.CrossRefGoogle Scholar
  26. 26.
    Pollegioni L, Sacchi S, Caldinelli L et al. (2007) Engineering the properties of D-amino acid oxidases by a rational and a directed evolution approach. Curr Protein Pept Sci 8, 600–618.PubMedCrossRefGoogle Scholar
  27. 27.
    Molla G, Sacchi S, Bernasconi M et al. (2006) Characterization of human D-amino acid oxidase. FEBS Lett 580, 2358–2364.PubMedCrossRefGoogle Scholar
  28. 28.
    Pollegioni L, Caldinelli L, Molla G et al. (2004) Catalytic properties of D-amino acid oxidase in cephalosporin C bioconversion: a comparison between proteins from different sources. Biotechnol Prog 20, 467–473.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Gianluca Molla
    • 1
    • 2
    Email author
  • Luciano Piubelli
    • 1
    • 2
  • Federica Volontè
    • 1
    • 2
  • Mirella S. Pilone
    • 1
    • 2
  1. 1.Dipartimento di Biotecnologie e Scienze MolecolariUniversità degli Studi dell’InsubriaVareseItaly
  2. 2.“The Protein Factory”Politecnico di Milano and Università degli Studi dell’InsubriaVareseItaly

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