title: Bringing nitrilase sequences from databases to life: the search for novel substrate specificities with a focus on dinitriles.
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.
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.|Department of Biochemistry, Charles University in Prague, Hlavova 8, CZ-128 40, Prague, Czech Republic.
authors.forename: Alicja B
authors.initials: AB
authors.lastname: Veselá
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Rucká
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.|Institute of Macromolecular Chemistry, Czech Academy of Sciences, Heyrovského nám. 2, CZ-162 06, Prague, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Kaplan
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Helena
authors.initials: H
authors.lastname: Pelantová
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Nešvera
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Pátek
authors.affiliation: Institute of Microbiology, Czech Academy of Sciences, Vídeňská 1083, CZ-142 20, Prague, Czech Republic. martinko@biomed.cas.cz.
authors.forename: Ludmila
authors.initials: L
authors.lastname: Martínková
mesh_terms: D000619:Aminohydrolases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D019295:Computational Biology; D057225:Data Mining; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D009570:Nitriles / Q000378:metabolism*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D009570:Nitriles; D011994:Recombinant Proteins; D000619:Aminohydrolases; C021867:nitrilase
keywords: Arylacetonitrilases; Cyano acids; Dinitriles; Genome mining; Nitrilases