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Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles

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Abstract

The aim of this study was to discover new nitrilases with useful activities, especially towards dinitriles that are precursors of high-value cyano acids. Genes coding for putative nitrilases of different origins (fungal, plant, or bacterial) with moderate similarities to known nitrilases were selected by mining the GenBank database, synthesized artificially and expressed in Escherichia coli. The enzymes were purified, examined for their substrate specificities, and classified into subtypes (aromatic nitrilase, arylacetonitrilase, aliphatic nitrilase, cyanide hydratase) which were largely in accordance with those predicted from bioinformatic analysis. The catalytic potential of the nitrilases for dinitriles was examined with cyanophenyl acetonitriles, phenylenediacetonitriles, and fumaronitrile. The nitrilase activities and selectivities for dinitriles and the reaction products (cyano acid, cyano amide, diacid) depended on the enzyme subtype. At a preparative scale, all the examined dinitriles were hydrolyzed into cyano acids and fumaronitrile was converted to cyano amide using E. coli cells producing arylacetonitrilases and an aromatic nitrilase, respectively.

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Funding

This study was funded by the Czech Science Foundation (No. P504/11/034), the Technology Agency of the Czech Republic (grant no. TA01021368), and the Institute of Microbiology of the Czech Academy of Sciences (No. RVO61388971).

Conflict of interest

The authors declare that they have no competing interests.

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This article does not contain any studies with human participants or animals performed by any of the authors.

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Authors and Affiliations

  1. Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic

    Alicja B. Veselá, Lenka Rucká, Ondřej Kaplan, Helena Pelantová, Jan Nešvera, Miroslav Pátek & Ludmila Martínková

  2. Department of Biochemistry, Charles University in Prague, Hlavova 8, CZ-128 40, Prague, Czech Republic

    Alicja B. Veselá

  3. Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, CZ-162 06, Prague, Czech Republic

    Ondřej Kaplan

Authors
  1. Alicja B. Veselá
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  2. Lenka Rucká
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  3. Ondřej Kaplan
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  4. Helena Pelantová
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  5. Jan Nešvera
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  6. Miroslav Pátek
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  7. Ludmila Martínková
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Correspondence to Ludmila Martínková.

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Alicja B. Veselá and Lenka Rucká contributed equally to this work.

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Veselá, A.B., Rucká, L., Kaplan, O. et al. Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles. Appl Microbiol Biotechnol 100, 2193–2202 (2016). https://doi.org/10.1007/s00253-015-7023-1

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