Advertisement

Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Genome mining of cyanide-degrading nitrilases from filamentous fungi

Abstract

A variety of fungal species are known to degrade cyanide through the action of cyanide hydratases, a specialized subset of nitrilases which hydrolyze cyanide to formamide. In this paper, we report on two previously unknown and uncharacterized cyanide hydratases from Neurospora crassa and Aspergillus nidulans. Recombinant forms of four cyanide hydratases from N. crassa, A. nidulans, Gibberella zeae, and Gloeocercospora sorghi were prepared after their genes were cloned with N-terminal hexahistidine purification tags, expressed in Escherichia coli, and purified using immobilized metal affinity chromatography. These enzymes were compared according to their relative specific activity, pH activity profiles, thermal stability, and ability to remediate cyanide contaminated waste water from silver and copper electroplating baths. Although all four were similar, the N. crassa cyanide hydratase (CHT) has the greatest thermal stability and widest pH range of >50% activity. N. crassa also demonstrated the highest rate of cyanide degradation in the presence of both heavy metals. The CHT of A. nidulans has the highest reaction rate of the four fungal nitrilases evaluated in this work. These data will help determine optimization procedures for the possible use of these enzymes in the bioremediation of cyanide-containing waste. Similar to known plant pathogenic fungi, both N. crassa and A. nidulans were induced to express CHT by growth in the presence of KCN.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4

References

  1. Akcil A, Mudder T (2003) Microbial destruction of cyanide wastes in gold mining: process review. Biotechnol Lett 25:445–450

  2. Banerjee A, Sharma R, Banerjee UC (2002) The nitrile-degrading enzymes: current status and future prospects. Appl Microbiol Biotechnol 60:33–44

  3. Barclay M, Tett VA, Knowles CJ (1998a) Metabolism and enzymology of cyanide/metallocyanide biodegradation by Fusarium solani under neutral and acidic conditions. Enzyme Microb Technol 23:321–330

  4. Baxter J, Cummings SP (2006) The current and future applications of microorganism in the bioremediation of cyanide contamination. Antonie Van Leeuwenhoek 90:1–17

  5. Brenner C (2002) Catalysis in the nitrilase superfamily. Curr Opin Struct Biol 12:775–782

  6. Cluness MJ, Turner PD, Clements E, Brown DT, O'Reilly C (1993) Purification and properties of cyanide hydratase from Fusarium lateritium and analysis of the corresponding chy1 gene. J Gen Microbiol 139:1807–1815

  7. Fisher FB, Brown JS (1952) Colorimetric determination of cyanide in stack gas and waste water. Analyt Chem 24:1440–1444

  8. Hardy RW, Knight E Jr. (1967) ATP-dependent reduction of azide and HCN by N2-fixing enzymes of Azotobacter vinelandii and Clostridium pasteurianum. Biochim Biophys Acta 139:69–90

  9. Ingvorsen K, Hojer-Pedersen B, Godtfredsen SE (1991) Novel cyanide-hydrolyzing enzyme from Alcaligenes xylosoxidans subsp. denitrificans.. Appl Environ Microbiol 57:1783–1789

  10. Jandhyala D (2002) Cyanide degrading nitrilases for detoxification of cyanide containing waste watersBiology and Biochemistry. University of Houston, Houston, p 130

  11. Jandhyala DM, Willson RC, Sewell BT, Benedik MJ (2005) Comparison of cyanide-degrading nitrilases. Appl Microbiol Biotechnol 68:327–335

  12. Kaminskyj SGW (2001) Fundamentals of growth, storage, genetics, and microscopy of Aspergillus nidulans. Fungal Genet Newsl 48:25–31

  13. Kobayashi M, Shimizu S (1994) Versatile nitrilases: nitrile-hydrolysing enzymes. FEMS Microbiol Lett 120:217–224

  14. Kobayashi M, Goda M, Shimizu S (1998) Nitrilase catalyzes amide hydrolysis as well as nitrile hydrolysis. Biochem Biophys Res Commun 253:662–666

  15. Li J, Burgess BK, Corbin JL (1982) Nitrogenase reactivity: cyanide as substrate and inhibitor. Biochem 21:4393–4402

  16. Mathew CD, Nagasawa T, Kobayashi M, Yamada H (1988) Nitrilase-catalyzed production of nicotinic acid from 3-cyanopyridine in Rhodococcus rhodochrous J1. Appl Environ Microbiol 54:1030–1032

  17. McCluskey K (2003) The Fungal Genetics Stock Center: from molds to molecules. Adv Appl Microbiol 52:245–262

  18. Meyers PR, Rawlings DE, Woods DR, Lindsey GG (1993) Isolation and characterization of a cyanide dihydratase from Bacillus pumilus C1. J Bacteriol 175:6105–6112

  19. Miller MD, Benedik MJ, Sullivan MC, Shipley NS, Krause KL (1991) Crystallization and preliminary crystallographic analysis of a novel nuclease from Serratia marcescens. J Mol Biol 222:27–30

  20. Nolan LM, Harnedy PA, Turner P, Hearne AB, O'Reilly C (2003) The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity. FEMS Microbiol Lett 221:161–165

  21. Pace HC, Brenner C (2001) The nitrilase superfamily: classification, structure and function. Genome Biol 2:REVIEWS0001

  22. Pertsovich SI, Guranda DT, Podchernyaev DA, Yanenko AS, Svedas VK (2005) Aliphatic amidase from Rhodococcus rhodochrous M8 is related to the nitrilase/cyanide hydratase family. Biochem 70:1280–1287

  23. Piotrowski M, Schonfelder S, Weiler EW (2001) The Arabidopsis thaliana isogene NIT4 and its orthologs in tobacco encode beta-cyano-l-alanine hydratase/nitrilase. J Biol Chem 276:2616–2621

  24. Sexton AC, Howlett BJ (2000) Characterisation of a cyanide hydratase gene in the phytopathogenic fungus Leptosphaeria maculans. Mol Gen Genet 263:463–470

  25. Studier FW (2005) Protein production by auto-induction in high density shaking cultures. Protein Expr Purif 41:207–234

  26. Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microbiol Genet Bull 13:42–43

  27. Wang P, VanEtten HD (1992) Cloning and properties of a cyanide hydratase gene from the phytopathogenic fungus Gloeocercospora sorghi. Biochem Biophys Res Commun 187:1048–1054

  28. Wang P, Matthews DE, VanEtten HD (1992) Purification and characterization of cyanide hydratase from the phytopathogenic fungus Gloeocercospora sorghi. Arch Biochem Biophys 298:569–575

  29. Watanabe A, Yano K, Ikebukuro K, Karube I (1998a) Cloning and expression of a gene encoding cyanidase from Pseudomonas stutzeri AK61. Appl Microbiol Biotechnol 50:93–97

  30. Watanabe A, Yano K, Ikebukuro K, Karube I (1998b) Cyanide hydrolysis in a cyanide-degrading bacterium, Pseudomonas stutzeri AK61, by cyanidase. Microbiology 144(Pt 6):1677–1682

  31. Westley J (1987) Thiosulfate: cyanide sulfurtransferase (rhodanese). Methods Enzymol 77:285–291

  32. Yanase H, Sakamoto A, Okamoto K, Kita K, Sato Y (2000) Degradation of the metal-cyano complex tetracyanonickelate (II) by Fusarium oxysporum N-10. Appl Microbiol Biotechnol 53:328–334

Download references

Acknowledgments

The financial support of the Robert A. Welch Society (Grants E-1263 and A-1310) and the Texas Hazardous Substance Research Center are gratefully acknowledged. The Fungal Genetic Stock Center created the locus disruptions promptly upon our request, and Dr. Wayne Versaw was instrumental in assisting us with the growth and characterization of the fungal strains.

Author information

Correspondence to Michael J. Benedik.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Basile, L.J., Willson, R.C., Sewell, B.T. et al. Genome mining of cyanide-degrading nitrilases from filamentous fungi. Appl Microbiol Biotechnol 80, 427 (2008). https://doi.org/10.1007/s00253-008-1559-2

Download citation

Keywords

  • Cyanide
  • Cyanide hydratase
  • Bioremediation