Functional Expression and Characterization of a Panel of Cobalt and Iron-Dependent Nitrile Hydratases

Birgit Grill, Maximilian Glänzer, Helmut Schwab, Steiner Kerstin, Daniel Pienaar, Dean Brady, Kai Donsbach, Margit Winkler*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


Nitrile hydratases (NHase) catalyze the hydration of nitriles to the corresponding amides. We report on the heterologous expression of various nitrile hydratases. Some of these enzymes have been investigated by others and us before, but sixteen target proteins represent novel sequences. Of 21 target sequences, 4 iron and 16 cobalt containing proteins were functionally expressed from Escherichia coli BL21 (DE3) Gold. Cell free extracts were used for activity profiling and basic characterization of the NHases using the typical NHase substrate methacrylonitrile. Co-type NHases are more tolerant to high pH than Fe-type NHases. A screening for activity on three structurally diverse nitriles was carried out. Two novel Co-dependent NHases from Afipia broomeae and Roseobacter sp. and a new Fe-type NHase from Gordonia hydrophobica were very well expressed and hydrated methacrylonitrile, pyrazine-carbonitrile, and 3-amino-3-(p-toluoyl)propanenitrile. The Co-dependent NHases from Caballeronia jiangsuensis and Microvirga lotononidis, as well as two Fe-dependent NHases from Pseudomonades, were—in addition—able to produce the amide from cinnamonitrile. Summarizing, seven so far uncharacterized NHases are described to be promising biocatalysts.
Original languageEnglish
Article number2521
Number of pages11
Issue number11
Publication statusPublished - 28 May 2020


  • active pharmaceutical ingredient.
  • amide
  • biocatalysis
  • metalloprotein
  • nitrile
  • nitrile hydratase (NHase)
  • non-corrinoid cobalt
  • non-heme iron

ASJC Scopus subject areas

  • Drug Discovery
  • Analytical Chemistry
  • Chemistry (miscellaneous)
  • Molecular Medicine
  • Physical and Theoretical Chemistry
  • Pharmaceutical Science
  • Organic Chemistry

Fields of Expertise

  • Human- & Biotechnology


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