Enzymatic Production of D-Amino Acids

  • Hemraj S. Nandanwar
  • Gurinder S. Hoondal
  • Rakesh M. Vohra
Part of the Methods in Biotechnology book series (MIBT, volume 17)


Optically pure amino acids are of increasing industrial importance as chiral building blocks for the synthesis of food ingredients, pharmaceuticals, drugs, and drug intermediates. Highly stereoselective enzymatic processes have been developed to obtain either D- or L-amino acids from D,L-mono-substituted hydantoin derivatives. The initial enzymatic reaction step of D,L-mono-substituted hydantoin hydrolysis is catalyzed by a D-hydantoinase after the subsequent racemization of L-isomer to D-isomer and leads to N-carbamoyl-D-amino acid. It is further hydrolyzed to D-amino acid by D-carbamoylase. Agrobacterium radiobacter and some other Agrobacterium species discussed in this chapter posses both the enzymes viz. D-hydantoinase and D-carbamoylase. In this chapter, D-p-hydroxyphenylglycine production from D,L-p-hydrox-yphenyl hydantoin has been studied in more detail. The purpose of this chapter is to provide the comprehensive idea to the readers directly from producing biocatalyst, analytical methods, harvesting the D-amino acids, and its characterization.

Key Words

D-hydantoinase L-carbamoylase Agrobacterium radiobacter biocatalysts D,L-p-hydroxyphenylhydantoin D-p-hydroxyphenylglycine 


  1. 1.
    Chibata, I. (1980) Development of enzyme-engineering-application of immobilized cell system, in Food Process Engineering, Vol. 2 (Linko, P. and Larinkari, J., eds.), Appl. Sci. Publishers, London, pp. 1–39.Google Scholar
  2. 2.
    Ariens, E. J. (1986) Stereochemistry: A source of problem in medicinal chemistry (part 1). Med. Res. Rev. 6, 451–466.PubMedCrossRefGoogle Scholar
  3. 3.
    Margolin, A. L. (1993) Enzymes in the synthesis of chiral drugs. Enzyme Microb. Technol. 15, 226–280.CrossRefGoogle Scholar
  4. 4.
    Louwrier, E. and Knowles, C. J. (1996) The purification and characterization of a novel D(-)-specific carbamoylase enzyme from an Agrobacterium sp. Enzyme. Microb. Technol. 19, 562–571.CrossRefGoogle Scholar
  5. 5.
    Lee, D. C., Lee, S. G., and Kim, H. S. (1996) Production of D-p-hydroxyphenyl-glycine from DL-5-4l-hydroxyphenylhydantoin using immobilized thermostable D-hydantoinase from Bacillus stearothermophillus. SD1. Enzyme Microb. Technol. 18, 35–40.CrossRefGoogle Scholar
  6. 6.
    Boer, L. de and Dijkhnizen, L. (1990) Microbial and enzymatic process for L-phenylalanine, in Advances in Biochemical Engineering/Biotechnology (Fiechter, A., ed.), Springer, Berlin, pp. 2–3.Google Scholar
  7. 7.
    Rhem, H. J. and Reed, G. (1996) Amino acids. Technical production and use, in Biotechnology (Puhler, A. and Stadler, P., eds.), VCH-Verlag, Weinheim, Germany, 6a, pp. 466–502.CrossRefGoogle Scholar
  8. 8.
    Yokozeki, K., Nakamori, S., Eguchi, C., Yamada, K., and Mitsugi, K. (1987) Screening of microorganisms producing D-p-hydroxyphenylglycine from D,L-5-(p-hydroxyphenyl)hydantoin. Agric. Biol. Chem. 51, 355–362.Google Scholar
  9. 9.
    Oliveri, R., Fascetti, E., Angelini, I., and Degen, L. (1981) Microbial transformation of racemic hydantoins to D-amino acids. Biotechnol. Bioeng. 23, 2173–2183.CrossRefGoogle Scholar
  10. 10.
    Moller, A., Syldatk, C., Schulze, M., and Wagner, F. (1988) Stereo-and substrate-specificity of a D-hydantoinase and a D-N-carbamoyl amino acid amidohydrolase of Arthrobacter crystallopoietes AM2. Enzyme Microb. Technol. 10, 618–625.CrossRefGoogle Scholar
  11. 11.
    Runser, S., Chinski, N., and Ohleyer, E. (1990) D-p-hydroxyphenylglycine production from D,L-5-hydroxyphenylhydantoin by Agrobacterium sp. Appl. Microbiol. Biotechnol. 33, 382–388.CrossRefGoogle Scholar
  12. 12.
    Sharma, R. and Vohra, R. M. (1997) A thermostable D-hydantoinase isolated from a mesophilic Bacillus sp. AR9. Biochem. Biophy. Res. Comm. 234, 485–488.CrossRefGoogle Scholar
  13. 13.
    Sarin, D., Sharma, R., Nandanwar, H. S., and Vohra, R. M. (2001) Two-step purification of D(-)-specific carbamoylase from Agrobacterium tumefaciens AM10. Protein Expres. Purif. 21, 170–175.CrossRefGoogle Scholar
  14. 14.
    Chao, Y. P., Juang, T. Y., Chern, J. T., and Lee, C. K. (1999a) Production of D-p-hydroxyphenylglycine by N-carbamoyl-D-amino acid amidohydrolase-overpro-ducing Escherichia coli strains. Biotechnol. Prog. 15, 603–607.PubMedCrossRefGoogle Scholar
  15. 15.
    Deepa, S., Sivasankar, B., Jayararman, K., Prabhakaran, K., George, S., Palani, P., et al. (1993) Enzymatic production and isolation of D-amino acids from the corresponding 5-substituted hydantoins. Process Biochem. 28, 447–452.CrossRefGoogle Scholar
  16. 16.
    Chao, Y. P., Fu, H., Lo, T. E., Chen, P. T., and Wang, J. J. (1999b) One-step production of D-P-hydroxyphenylglycine by recombinant Escherichia coli strains. Biotechnol. Prog. 15, 1039–1045.PubMedCrossRefGoogle Scholar
  17. 17.
    Buson, A., Negro, A., Grassato, L., Tagliaro, M., Basaglia, M., Grandi, C., et al. (1996) Identification, sequencing and mutagenesis of the gene for a D-carbamoy-lase from Agrobacterium radiobacter. FEMS Microbiol. Lett. 145, 55–62.PubMedGoogle Scholar
  18. 18.
    Runser, S., Chinski, N., and Ohleyer, E. (1990) D-p-hydroxyphenylglycine production from D,L-p-hydroxyphenylhydantoin by Agrobacterium species. Appl. Microbiol. Biotechnol. 33, 382–388.CrossRefGoogle Scholar
  19. 19.
    Meyer, P. and Runser, S. (1993) Efficient production of the industrial biocatalysts hydantoinase and N-carbamoylamino acid amidohydrolase: novel non-metaboliz-able inducers. FEMS Microbiol. Lett. 109, 67–74.CrossRefGoogle Scholar
  20. 20.
    Park, H. H., Kim, G. J., and Kim, H. S. (2000) Production of D-amino acids using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase. Biotechnol. Prog. 16, 564–570.PubMedCrossRefGoogle Scholar
  21. 21.
    Ley, C. J., Kirchmann, S., Burton, S. G., and Dorrington, R. (1998) Production of D-amino acids from D,L-5-substituted hydantoins by an Agrobacterium tumefa-ciens strain and isolation of a mutant with inducer-independent expression of hydantoin-hydrolysing activity. Biotechnol. Lett. 20, 707–711.CrossRefGoogle Scholar
  22. 22.
    Hsu, W. H., Chien, F. T., Hsu, C. L., Wang, T. C., Yuan, H. S., and Wang, W. C. (1999) Expression, crystallization, and preliminary X-ray diffraction studies of N-carbamoyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter. Acta Crystallogr D D55, 694–695.CrossRefGoogle Scholar
  23. 23.
    Morin, A., Hummel, W., and Kula, M. R. (1986) Rapid detection of microbial hydantoinase on solid medium. Biotechnol Lett. 8, 573–576.CrossRefGoogle Scholar
  24. 24.
    Nakai, T., Hasegawa, T., Yamashita, E., Yamamoto, M., Kumasaka, T., Ueki, T., et al. (2000) Crystal structure of N-carbamoyl-D-amino acid amidohydrolase with a novel catalytic framework common to amidohydrolases. Structure Fold, Des. 15, 729–737.CrossRefGoogle Scholar
  25. 25.
    Nanba, H., Ikenaka, Y., Yamada, Y., Yajima, K., Takano, M., and Takahashi, S. (1998) Isolation of Agrobacterium sp. KNK712 that produces N-carbamoyl-D-amino acid amidohydrolase, cloning of the gene for this enzyme, and properties of the enzyme. Biosci. Biotech. Biochem. 62, 875–881.CrossRefGoogle Scholar
  26. 26.
    Ohasi, T., Takahashi, S., Nagamachi, T., and Yoneda, H. (1981) A new method for 5-(4-hydroxyphenyl)-hydantoin synthesis. Agric. Biol. Chem. 45, 831–838.Google Scholar
  27. 27.
    Henze, H. R. and Speer, R. J. (1942) Identification of carbamoyl compounds through conversion into hydantoin. J. Am. Chem. Soc. 64, 522–523.CrossRefGoogle Scholar
  28. 28.
    Suzuki, T., Igarashi, K., Hase, K., and Tuzimura, K. (1973): Optical rotatory dispersion and circular dichorism of amino acid hydantoin. Agric. Biol. Chem. 37, 411–416.Google Scholar
  29. 29.
    Yamada, S., Hongo, C., and Chibata, I. (1978) Preparation of D-p-hydroxyphenyl-glycine: optical resolution of D,L-p-hydroxyphenylglycine by preferential crystallization procedure. Agric. Biol. Chem. 42, 1521–1526.Google Scholar
  30. 30.
    Takahashi, S., Ohashi, T., Kii, Y., Kumagai, H., and Yamada, H. (1979) Microbial transformation of hydantoins to N-carbamoyl-D-amino acids. J. Ferment. Technol. 57, 328–332.Google Scholar

Copyright information

© Humana Press Inc. 2005

Authors and Affiliations

  • Hemraj S. Nandanwar
    • 1
  • Gurinder S. Hoondal
    • 2
  • Rakesh M. Vohra
    • 2
  1. 1.Institute of Microbial TechnologyChandigarhIndia
  2. 2.Department of MicrobiologyPanjab UniversityChandigarhIndia

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