Skip to main content

Thank you for visiting nature.com. 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.

  • Letter
  • Published:

Enantioseparation using apoenzymes immobilized in a porous polymeric membrane

Nature volume 388, pages 758–760 (1997)Cite this article

Abstract

Chemical separations represent a large portion of the cost of bringing any new pharmaceutical product to the market. Membrane-based separation technologies1,2, in which the target molecule is selectively extracted and transported across a membrane, are potentially more economical and easier to implement than competing separations methods; but membranes with higher transport selectivities are required. Here we describe an approach for preparing highly selective membranes which involves immobilizing apoenzymes within a microporous composite. The apoenzyme selectively recognizes its substrate molecule and transports it across the composite membrane, without effecting the unwanted chemical conversion of the substrate molecule to product. We demonstrate this approach using three different apoenzymes. Most importantly, it can be used to make enantioselective membranes for chiral separations, one of the most challenging and important problems in bioseparations technology. We are able to achieve a fivefold difference between the transport rates of D- and L-amino acids.

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

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

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

Figure 1: Schematic cross-section of the polypyrrole/polycarbonate/polypyrrole sandwich membrane with the apoenzyme entrapped in the pores.
Figure 2: a, Plots of amount of ethanol transported from the feed solution through the membrane and into the permeant solution versus time for a membrane loaded with apo-ADH and for an apo-ADH-free membrane.
Figure 3: a, Plots of amount of ethanol transported from the feed solution through the membrane and into the permeant solution versus time for a membrane loaded with apo-ADH and for an apo-ADH-free membrane.
Figure 4: a, Plot of ethanol and phenol flux versus concentration of ethanol and phenol in the feed solution for an apo-ADH-loaded membrane.
Figure 5: a, Plot of ethanol and phenol flux versus concentration of ethanol and phenol in the feed solution for an apo-ADH-loaded membrane.
Figure 6: a, Plots of D-phenylalanine and L-phenylalanine flux versus concentration of these enantiomers in the feed solution for an apo-D-AAO-loaded membrane.
Figure 7: a, Plots of D-phenylalanine and L-phenylalanine flux versus concentration of these enantiomers in the feed solution for an apo-D-AAO-loaded membrane.

Similar content being viewed by others

References

  1. Baker, R. W.et al. Membrane Separation Systems(Noyes Data Corp., New Jersey, (1991).

    Google Scholar 

  2. Winston, W. H. & Sirkar, K. K. Membrane Handbook(Van Nostrand Reinhold, New York, (1992)).

    Google Scholar 

  3. Stein, W. D. The Movement of Molecules Across Cell Membranes(Academic, New York, (1967)).

    Google Scholar 

  4. Scholander, P. F. Oxygen transport through haemoglobin solutions. Science 131, 585–590 (1960).

    Article  ADS  CAS  Google Scholar 

  5. Chen, X., Nishide, H. & Tsuchida, E. Analysis of facilitated oxygen transport in a liquid membrane of haemoglobin. Bull. Chem. Soc. Jpn 69, 255–259 (1996).

    Article  CAS  Google Scholar 

  6. Rethwisch, D. G., Yi, G., Subramanian, A. & Dordick, J. S. Enzyme-facilitated transport and separation of organic acids through liquid membranes. J. Am. Chem. Soc. 112, 1649–1650 (1990).

    Article  CAS  Google Scholar 

  7. Rethwisch, D. G., Parida, S., Yi, G. & Dordick, J. S. Use of alcohols as cosolvents in enzyme-facilitated transport of organic acids through liquid membranes. J. Membr. Sci. 95, 83–91 (1994).

    Article  CAS  Google Scholar 

  8. Dordick, J. S. Biocatalysts in Industry 311–325 (Plenum, New York, (1989)).

    Google Scholar 

  9. Stanley, T. J. & Quinn, J. A. Phase transfer catalysis in a membrane reactor. Chem. Eng. Sci. 42, 2313–2324 (1987).

    Article  CAS  Google Scholar 

  10. Matson, S. L. & Lopez, J. L. Frontiers in Bioprocessing(CRC, Boca Raton, (1989)).

    Google Scholar 

  11. Parthasarathy, R. V. & Martin, C. R. Synthesis of polymeric microcapsule arrays and their use for enzyme immobilization. Nature 369, 298–301 (1994).

    Article  ADS  CAS  Google Scholar 

  12. Nishizawa, M., Menon, V. P. & Martin, C. R. Metal nanotubule membrane with electrochemically switchable ion-transport selectivity. Science 268, 700–702 (1995).

    Article  ADS  CAS  Google Scholar 

  13. Martin, C. R. Nanomaterials: A membrane-based synthetic approach. Science 266, 1961–1966 (1994).

    Article  ADS  CAS  Google Scholar 

  14. Martin, C. R. Membrane-based synthesis of nanomaterials. Chem. Mater. 8, 1739–1746 (1996).

    Article  CAS  Google Scholar 

  15. Penner, R. M., Van Dyke, L. S. & Martin, C. R. Electrochemical evaluation of charge transport rates in polypyrrole. J. Phys. Chem. 92, 5274–5282 (1988).

    Article  CAS  Google Scholar 

  16. Noble, R. D., Koval, C. A. & Pellegrino, J. J. Facilitated transport membrane systems. Chem. Eng. Prog. 85, 58–70 (1989).

    CAS  Google Scholar 

  17. Noble, R. D. Generalized microscopic mechanism of faciitated transport in fixed carrier membranes. J. Membr. Sci. 75, 121–129 (1992).

    Article  CAS  Google Scholar 

  18. Lakshmi, B. B. Unpublished results.

  19. Borman, S. Chirality emerges as key issue in pharmaceutical research. Chem. Eng. News 68, 9–12 (1990).

    Article  Google Scholar 

  20. Stinson, S. C. Chiral drugs. Chem. Eng. News 73, 44 (1995).

    Article  Google Scholar 

  21. Collins, A. N., Sheldrake, G. N. & Crosby, J. Chirality in Industry(Wiley & Sons, Chichester, (1992)).

    Google Scholar 

  22. Dixon, M. & Kleppe, K. D-amino acid oxidase-specificity, competititve inhibition and reaction sequence. Biochim. Biophys. Acta 96, 368–382 (1965).

    Article  CAS  Google Scholar 

  23. Higuchi, A., Ishida, Y. & Nakagawa, T. Surface modified polysulfone membranes: separation of mixed proteins and optical resolution of tryptophan. Desalination 90, 127–136 (1993).

    Article  CAS  Google Scholar 

  24. Higuchi, A., Hara, M., Horiuchi, T. & Nakagawa, T. Optical resolution of amino acids by ultrafiltration membranes containing serum albumin. J. Membr. Sci. 93, 157–164 (1994).

    Article  CAS  Google Scholar 

  25. Yoshikawa, M., Izumi, J., Kitao, T., Koya, S. & Sakamoto, S. Molecularly imprinted polymeric membranes for optical resolution. J. Membr. Sci. 108, 171–175 (1995).

    Article  CAS  Google Scholar 

  26. Maruyama, A.et al. Enantioselective permeation of α-amino acid isomers through poly(amino acid) derived membranes. Macromolecules 23, 2748–2752 (1990).

    Article  ADS  CAS  Google Scholar 

  27. Kakuchi, T., Takaoka, T. & Yokota, K. Polymeric chiral crown ethers VI. Optical resolution of α-amino acid by polymers incorporating 1,3,4,6-di-O-benzylidine-D-mannitol residues. Polym. J. 22, 199–205 (1990).

    Article  CAS  Google Scholar 

  28. Parthasarathy, A., Brumlik, C. J., Martin, C. R. & Collins, G. E. Interfacial polymerization of thin polymer films onto the surface of a microporous hollow fiber membrane. J. Membr. Sci. 94, 249–254 (1994).

    Article  CAS  Google Scholar 

  29. Jensen, R. D., Gill, S. C., Pardi, A. & Polisky, B. High resolution molecular discrimination by RNA. Science 263, 1425–1429 (1994).

    Article  ADS  Google Scholar 

  30. Boyer, P. D. The Enzymes(Academic, New York, (1975)).

    Google Scholar 

  31. Worthington, C. C. Worthington Manual(Worthington Biochemical Corp., New Jersey, (1988)).

    Google Scholar 

Download references

Acknowledgements

This work was supported by the US NSF and the US Office of Naval Research. We thank C. A. Koval for discussions.

Author information

Authors and Affiliations

  1. Department of Chemistry, Colorado State University, Fort Collins, 80523, Colorado, USA

    Brinda B. Lakshmi & Charles R. Martin

Author notes

  1. Correspondence and requests for materials should be addressed to C.R.M.

    Authors
    1. Brinda B. Lakshmi
      View author publications

      You can also search for this author in PubMed Google Scholar

    2. Charles R. Martin
      View author publications

      You can also search for this author in PubMed Google Scholar

    Rights and permissions

    Reprints and permissions

    About this article

    Cite this article

    Lakshmi, B., Martin, C. Enantioseparation using apoenzymes immobilized in a porous polymeric membrane. Nature 388, 758–760 (1997). https://doi.org/10.1038/41978

    Download citation

    • Received:

    • Accepted:

    • Issue Date:

    • DOI: https://doi.org/10.1038/41978

    This article is cited by

    Comments

    By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

    Search

    Advanced search

    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