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Applied Microbiology and Biotechnology

, Volume 100, Issue 5, pp 2193–2202 | Cite as

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

  • Alicja B. Veselá
  • Lenka Rucká
  • Ondřej Kaplan
  • Helena Pelantová
  • Jan Nešvera
  • Miroslav Pátek
  • Ludmila Martínková
Biotechnologically relevant enzymes and proteins

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.

Keywords

Nitrilases Arylacetonitrilases Dinitriles Cyano acids Genome mining 

Notes

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.

Ethical approval

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

Supplementary material

253_2015_7023_MOESM1_ESM.pdf (279 kb)
ESM 1 (PDF 279 kb)

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Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  • Alicja B. Veselá
    • 1
    • 2
  • Lenka Rucká
    • 1
  • Ondřej Kaplan
    • 1
    • 3
  • Helena Pelantová
    • 1
  • Jan Nešvera
    • 1
  • Miroslav Pátek
    • 1
  • Ludmila Martínková
    • 1
  1. 1.Institute of MicrobiologyCzech Academy of SciencesPragueCzech Republic
  2. 2.Department of BiochemistryCharles University in PraguePragueCzech Republic
  3. 3.Institute of Macromolecular ChemistryCzech Academy of SciencesPragueCzech Republic

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