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Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes

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Abstract

Bacterial amidases and nitrile hydratases can be used for the synthesis of various intermediates and products in the chemical and pharmaceutical industries and for the bioremediation of toxic pollutants. The aim of this study was to analyze the expression of the amidase and nitrile hydratase genes of Rhodococcus erythropolis and test the stereospecific nitrile hydratase and amidase activities on chiral cyanohydrins. The nucleotide sequences of the gene clusters containing the oxd (aldoxime dehydratase), ami (amidase), nha1, nha2 (subunits of the nitrile hydratase), nhr1, nhr2, nhr3 and nhr4 (putative regulatory proteins) genes of two R. erythropolis strains, A4 and CCM2595, were determined. All genes of both of the clusters are transcribed in the same direction. RT-PCR analysis, primer extension and promoter fusions with the gfp reporter gene showed that the ami, nha1 and nha2 genes of R. erythropolis A4 form an operon transcribed from the Pami promoter and an internal Pnha promoter. The activity of Pami was found to be weakly induced when the cells grew in the presence of acetonitrile, whereas the Pnha promoter was moderately induced by both the acetonitrile or acetamide used instead of the inorganic nitrogen source. However, R. erythropolis A4 cells showed no increase in amidase and nitrile hydratase activities in the presence of acetamide or acetonitrile in the medium. R. erythropolis A4 nitrile hydratase and amidase were found to be effective at hydrolysing cyanohydrins and 2-hydroxyamides, respectively.

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Acknowledgments

This work was supported by grant LC06010 from the Ministry of Education, Czech Republic, by Grant P504/11/0394 from the Czech Science Foundation and by the institutional research concept RVO 61388971. We thank prof. H. Griengl (Graz University of Technology, Graz, Austria) for kindly providing the cyanohydrins as well as useful advice on their biotransformation and analysis and D. Lukavská for excellent technical assistance.

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The authors declare that they have no conflict of interest.

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Authors and Affiliations

  1. Institute of Microbiology AS CR, v. v. i., Vídeňská 1083, 142 20, Prague 4, Czech Republic

    Lenka Rucká, Olga Volkova, Adam Pavlík, Ondřej Kaplan, Martin Kracík, Jan Nešvera, Ludmila Martínková & Miroslav Pátek

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  1. Lenka Rucká
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  2. Olga Volkova
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  3. Adam Pavlík
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  4. Ondřej Kaplan
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  7. Ludmila Martínková
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  8. Miroslav Pátek
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Correspondence to Jan Nešvera.

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Rucká, L., Volkova, O., Pavlík, A. et al. Expression control of nitrile hydratase and amidase genes in Rhodococcus erythropolis and substrate specificities of the enzymes. Antonie van Leeuwenhoek 105, 1179–1190 (2014). https://doi.org/10.1007/s10482-014-0179-3

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  • DOI: https://doi.org/10.1007/s10482-014-0179-3

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