Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine


Using directed evolution, we have improved the hydantoinase process for production of L-methionine (L-met) in Escherichia coli. This was accomplished by inverting the enantioselectivity and increasing the total activity of a key enzyme in a whole-cell catalyst. The selectivity of all known hydantoinases for D-5-(2-methylthioethyl)hydantoin (D-MTEH) over the L-enantiomer leads to the accumulation of intermediates and reduced productivity for the L-amino acid. We used random mutagenesis, saturation mutagenesis, and screening to convert the D-selective hydantoinase from Arthrobacter sp. DSM 9771 into an L-selective enzyme and increased its total activity fivefold. Whole E. coli cells expressing the evolved L-hydantoinase, an L-N-carbamoylase, and a hydantoin racemase produced 91 mM L-met from 100 mM D,L-MTEH in less than 2 h. The improved hydantoinase increased productivity fivefold for >90% conversion of the substrate. The accumulation of the unwanted intermediate D-carbamoyl-methionine was reduced fourfold compared to cells with the wild-type pathway. Highly D-selective hydantoinase mutants were also discovered. Enantioselective enzymes rapidly optimized by directed evolution and introduced into multienzyme pathways may lead to improved whole-cell catalysts for efficient production of chiral compounds.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it


Prices may be subject to local taxes which are calculated during checkout

Figure 1: Reactions and enzymes involved in the production of L-—amino acids from racemic hydantoins by the three-enzyme hydantoinase process.
Figure 2: Representative example of screening results: activities toward D- and L-MTEH of 4,000 clones from the first random mutant library.
Figure 3
Figure 4: Evolutionary progression and enantioselectivities of the various mutants.
Figure 5


  1. Stinson, S.C. Counting on chiral drugs. Chem. Eng. News 76, 83–104 (1998).

    Article  Google Scholar 

  2. Syldatk, C., Müller, R., Siemann, M. & Wagner, F. Microbial and enzymatic production of D-amino acids from DL-5-monosubstituted hydantoins. In Hydrolysis and formation of hydantoins in biocatalytic production of amino acids and derivatives. Biocatalytic production of amino acids and derivitives. (eds Rozzell, J.D. & Wagner, F.) 75–127 (Hanser Publisher, New York; 1992).

    Google Scholar 

  3. Syldatk, C., Müller, R., Pietzsch, M. & Wagner, F. Microbial and enzymatic production of L-amino acids from DL-5-monosubstituted hydantoins. In Hydrolysis and formation of hydantoins in biocatalytic production of amino acids and derivatives. (eds Rozzell, J.D. & Wagner, F.) 131–1176 (Hanser Publisher, New York; 1992).

    Google Scholar 

  4. Drauz, K. Chiral amino acids: a versatile tool in the synthesis of pharmaceuticals and fine chemicals. Chimia 51, 310–314 (1997).

    CAS  Google Scholar 

  5. Wagner, F., Hantke, B., Wagner; T., Drauz K. & Bommarius, A. Microorganism, use thereof and process for the production of L-alpha-amino acids. US 5714355 (1998).

    Google Scholar 

  6. May, O. et al. Substrate-dependent enantioselectivity of a novel hydantoinase from Arthrobacter aurescens DSM 3745: purification and characterization as a new member of cyclic amidases. J. Biotechnol. 61, 1–13 (1998).

    Article  CAS  Google Scholar 

  7. Wagner, T., Hantke, B. & Wagner, F. Production of L-methionine from D,L-5-(2-methylthioethyl)hydantoin by resting cells of a new mutant strain of Arthrobacter species DSM 7330. J. Biotechnol. 46, 63–69 (1996).

    Article  CAS  Google Scholar 

  8. Völkel, D. & Wagner, F. Reaction mechanism for the conversion of 5-monosubstituted hydantoins to enantiomerically pure L-amino acids. Ann. NY Acad. Sci. 750, 1–9 (1995).

    Article  Google Scholar 

  9. Arnold, F.H. & Moore, J.C. Optimizing industrial enzymes by directed evolution. Adv. Biochem. Eng. Biotechnol. 58, 1–14 (1997).

    CAS  PubMed  Google Scholar 

  10. Arnold, F.H. & Wintrode, P.L. Fermentation, biocatalysis, and bioseparation. In Encyclopedia of bioprocess technology (eds Flickinger, M.C. & Drew, S.W.) 971–987 (John Wiley & Sons, New York, NY; 1999).

    Google Scholar 

  11. Matcham, G.W. & Bowen, A.R.S. Biocatalysis for chiral intermediates: meeting commercial and technical challenges. CHIM. OGGI 14, 20–24 (1996).

    CAS  Google Scholar 

  12. Reetz, M.T., Zonta A., Schimossek K., Liebeton, K. & Jaeger, K.-E. Creation of enantioselective biocatalysts for organic chemistry by in vitro evolution. Angew. Chem. Int. Edn. Engl. 36, 2830–2832 (1998).

    Article  Google Scholar 

  13. Reetz, M.T. & Jaeger, K.-E. Superior biocatalysts by directed evolution. Top. Curr. Chem. 200, 31–57 (1999).

    Article  CAS  Google Scholar 

  14. Miyazaki, K. & Arnold, F.H. Exploring nonnatural evolutionary pathways by saturation mutagenesis: rapid improvement of protein function J. Mol. Evol. 49, 716–720 (1999).

    Article  CAS  Google Scholar 

  15. Handelsman, J., Rondon, M.R., Brady, S.F., Clardy, J. & Goodman, R.M. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5, R245–R249 (1998).

    Article  CAS  Google Scholar 

  16. Fersht, A. (ed.) Enzyme structure and mechanism. (W.H. Freeman and Co., New York, NY; 1985).

    Google Scholar 

  17. Wilms, B. et al. Cloning, nucleotide sequence and expression of a new L-N-carbamoylase gene from Arthrobacter aurescens DSM 3747 in E. coli. J. Biotechnol. 68, 101–113 (1999).

    Article  CAS  Google Scholar 

  18. Volff, J.-N., Eichenseer, C., Viell, P., Piendl, W. & Altenbuchner, J. Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplificable element AUDI of Streptomyces lividans 66. Mol. Microbiol. 21, 1037–1047 (1996).

    Article  CAS  Google Scholar 

  19. Rose, R.E. The nucleotide sequence of pACYC184. Nucleic Acids Res. 16, 355 (1988).

    Article  CAS  Google Scholar 

  20. Yanisch-Perron, C., Viera, J. & Messing, J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC vectors. Gene 33, 103–109 (1984).

    Article  Google Scholar 

  21. Luria, S.E., Adams, J.N. & Ting, R.C. Transduction of lactose-utilizing ability among strains of Escherichia coli and Shigella dysenteriae and properties of phage particles. Virology 12, 348–390 (1960).

    Article  CAS  Google Scholar 

  22. Chen, C.S., Fujimoto, Y., Girdaukas, G. & Sih, C.J. Quantitative analysis of biochemical kinetic resolutions of enantiomers. J. Am. Chem. Soc. 104, 7294–7299 (1982).

    Article  CAS  Google Scholar 

Download references


We thank Dr. Joe Altenbuchner and Dr. A. Wiese for kindly providing vector pJOE2702 and the racemase gene from Arthrobacter aurescens DSM3747, respectively, and Dr. W. Günther for his support in HPLC analytics. We also thank Dr. A. Bommarius for reading the manuscript and useful discussions and J. Ladd for his excellent technical assistance. This work was financed by Degussa-Huels AG. We also thank the Caltech SURF program for support of undergraduate research (P.N. and J. Ladd).

Author information

Authors and Affiliations


Corresponding author

Correspondence to Frances H. Arnold.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

May, O., Nguyen, P. & Arnold, F. Inverting enantioselectivity by directed evolution of hydantoinase for improved production of l-methionine. Nat Biotechnol 18, 317–320 (2000).

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

This article is cited by


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing