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.

  • Research Article
  • Published:

Directed evolution of a fungal peroxidase

Abstract

The Coprinus cinereus (CiP) heme peroxidase was subjected to multiple rounds of directed evolution in an effort to produce a mutant suitable for use as a dye-transfer inhibitor in laundry detergent. The wild-type peroxidase is rapidly inactivated under laundry conditions due to the high pH (10.5), high temperature (50°C), and high peroxide concentration (5–10 mM). Peroxidase mutants were initially generated using two parallel approaches: site-directed mutagenesis based on structure-function considerations, and error-prone PCR to create random mutations. Mutants were expressed in Saccharomyces cerevisiae and screened for improved stability by measuring residual activity after incubation under conditions mimicking those in a washing machine. Manually combining mutations from the site-directed and random approaches led to a mutant with 110 times the thermal stability and 2.8 times the oxidative stability of wild-type CiP. In the final two rounds, mutants were randomly recombined by using the efficient yeast homologous recombination system to shuffle point mutations among a large number of parents. This in vivo shuffling led to the most dramatic improvements in oxidative stability, yielding a mutant with 174 times the thermal stability and 100 times the oxidative stability of wild-type CiP.

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

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

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

Figure 1: Effect of single amino acid substitutions at position E239 on initial and residual activity.
Figure 2: Results of in vivo shuffling of improved mutants.
Figure 3: Evolution of oxidative stability.
Figure 4: Structural mapping of amino acid substitutions.

Similar content being viewed by others

References

  1. Nedwin, G.E. in Biotechnology in the Sustainable Environment. (eds. Sayler, G. et al). 13–32 (Plenum Press, New York, 1997 ).

    Book  Google Scholar 

  2. Kuchner, K. & Arnold, F.H. Directed evolution of enzyme catalysts. Trends Biotechnol. 15, 523– 530 (1997).

    Article  CAS  Google Scholar 

  3. Muller, H.J. The relation of recombination to mutational advance. Mutat. Res. 1, 2–9 (1964 ).

    Article  Google Scholar 

  4. Stemmer, W.P.C. 1994. DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution. Proc. Natl. Acad. Sci. USA 91, 10747–10751 ( 1964).

    Article  Google Scholar 

  5. Abelskov, A.K., Smith, A.T., Rasmussen, C.B., Dunford, H.B. & Welinder, K.G. pH-dependence and structural interpretation of the reactions of Coprinus cinereus peroxidase with hydrogen peroxide, ferulic acid, and 2,2´-azinobis (3-ethylbenzthiazoline-6-sulfonic acid). Biochemistry 36, 9453– 9463 (1997).

    Article  CAS  Google Scholar 

  6. Petersen, J.F.W. et al. Crystallization and x-ray-diffraction analysis of recombinant Coprinus cinereus peroxidase. J. Mol. Biol. 232 , 989–991 (1993).

    Article  CAS  Google Scholar 

  7. Kunishima, N., Fukuyama, K. & Matsubara, H. Crystal-structure of the fungal peroxidase from Arthromyces ramosus at 1.9 angstrom resolution - structural comparisons with the lignin and cytochrome-C peroxidases. J. Mol. Biol. 235, 331–344 (1994).

    Article  CAS  Google Scholar 

  8. Poulos, T.L. & Kraut, J. The stereochemistry of peroxidase catalysis. J. Biol. Chem. 255, 8199–8205 (1980).

    CAS  PubMed  Google Scholar 

  9. Lopez-Camacho, C. et al. J. Amino acid substitutions enhancing thermostability of Bacillus polymyxa beta-glucosidase A. Biochem. J. 314, 833–838 (1996).

    Article  CAS  Google Scholar 

  10. Matsumura, M., Yasumura, S. & Aiba, S. Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein. Nature 323, 356–358 (1986).

    Article  CAS  Google Scholar 

  11. Stemmer, W.P.C. Rapid evolution of a protein in vitro by DNA shuffling. Nature 370, 389–391 ( 1994).

    Article  CAS  Google Scholar 

  12. Zhao H.M. & Arnold F.H. Optimization of DNA shuffling for high-fidelity recombination. Nucleic. Acids Res. 25 , 1307–1308 (1997).

    Article  CAS  Google Scholar 

  13. Manivasakam, P., Weber, C.W., McElver, J. & Schiestl Micro-homology mediated PCR targeting in Saccharomyces cerevisiae. Nucleic Acids Res. 14, 2799–2800 ( 1995).

    Article  Google Scholar 

  14. Crameri, A., Raillard, S.A., Bermudez, E. & Stemmer, W.P.C. DNA shuffling of a family of genes from diverse species accelerates directed evolution. Nature 391, 288– 291 (1998).

    Article  CAS  Google Scholar 

  15. Cannon, J.F. & Tatchell, K. 1987. Characterization of Saccharomyces cerevisiae genes encoding subunits of cyclic AMP-dependent protein kinase. Molec. Cell. Biol. 7, 2653– 2663 (1987).

    Article  CAS  Google Scholar 

  16. Leung, D.E., Chen, E. & Goeddel, D.V.A. Method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique 1, 11–15 (1989).

    Google Scholar 

  17. Manivasakam, P. & Schiestl, R.H. High efficiency transformation of Saccharomyces cerevisiae by electroporation. Nucleic Acids Res. 21, 4414–4415 (1993).

    Article  CAS  Google Scholar 

  18. Pompon, D. & Nicolas, A. Protein engineering by cDNA recombination in yeasts: shuffling of mammalian cytochrome P-450 functions. Gene 83, 15–24 ( 1989).

    Article  CAS  Google Scholar 

  19. Landt, O., Grunert, H.P. & Hahn, U. A general method for rapid site-directed mutagenesis using the polymerase chain reaction. Gene 96, 125–128 (1990).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

The authors gratefully acknowlege the contributions of Juozas Kulys, Donna L. Moyer, Jeffery Shuster, and Birger Rostgaard Jensen.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Joel R. Cherry.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Cherry, J., Lamsa, M., Schneider, P. et al. Directed evolution of a fungal peroxidase. Nat Biotechnol 17, 379–384 (1999). https://doi.org/10.1038/7939

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/7939

This article is cited by

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