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Comparison of cyanide-degrading nitrilases

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Abstract

Recombinant forms of three cyanide-degrading nitrilases, CynD from Bacillus pumilus C1, CynD from Pseudomonas stutzeri, and CHT from Gloeocercospora sorghi, were prepared after their genes were cloned with C-terminal hexahistidine purification tags and expressed in Escherichia coli, and the enzymes purified using nickel-chelate affinity chromatography. The enzymes were compared with respect to their pH stability, thermostability, metal tolerance, and kinetic constants. The two bacterial genes, both cyanide dihydratases, were similar with respect to pH range, retaining greater than 50% activity between pH 5.2 and pH 8 and kinetic properties, having similar Km (6–7 mM) and Vmax (0.1 mmol min−1 mg−1). They also exhibited similar metal tolerances. However, the fungal CHT enzyme had notably higher Km (90 mM) and Vmax (4 mmol min−1 mg−1) values. Its pH range was slightly more alkaline (retaining nearly full activity above 8.5), but exhibited a lower thermal tolerance. CHT was less sensitive to Hg2+ and more sensitive to Pb2+ than the CynD enzymes. These data describe, in part, the current limits that exist for using nitrilases as agents in the bioremediation of cyanide-containing waste effluent, and may help serve to determine where and under what conditions these nitrilases may be applied.

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Acknowledgements

We gratefully acknowledge support from the Robert A. Welch Foundation, the University of Houston Environmental Institute, and the Gulf Coast Hazardous Substance Research Center, #069UHH0789, to M.J.B. and from the Wellcome Trust to B.T.S. for support of this project.

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Author notes

  1. Dakshina M. Jandhyala

    Present address: Institute of Veterinary, Animal, and Biomedical Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand

  2. Michael J. Benedik

    Present address: Department of Biology, Texas A&M University, College Station, TX, 77843-3258, USA

Authors and Affiliations

  1. Department of Biology and Biochemistry, University of Houston, Houston, TX, 77204-5001, USA

    Dakshina M. Jandhyala, Richard C. Willson & Michael J. Benedik

  2. Department of Chemical Engineering, University of Houston, Houston, TX, 77204-5001, USA

    Richard C. Willson

  3. Electron Microscope Unit, University of Cape Town, Rondebosch 7701, Cape Town, South Africa

    B. Trevor Sewell

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  1. Dakshina M. Jandhyala
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  2. Richard C. Willson
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  3. B. Trevor Sewell
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  4. Michael J. Benedik
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Correspondence to Michael J. Benedik.

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Jandhyala, D.M., Willson, R.C., Sewell, B.T. et al. Comparison of cyanide-degrading nitrilases. Appl Microbiol Biotechnol 68, 327–335 (2005). https://doi.org/10.1007/s00253-005-1903-8

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  • DOI: https://doi.org/10.1007/s00253-005-1903-8

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