title: Purification and characterization of a nitrilase from Aspergillus niger K10.
abstract: Aspergillus niger K10 cultivated on 2-cyanopyridine produced high levels of an intracellular nitrilase, which was partially purified (18.6-fold) with a 24% yield. The N-terminal amino acid sequence of the enzyme was highly homologous with that of a putative nitrilase from Aspergillus fumigatus Af293. The enzyme was copurified with two proteins, the N-terminal amino acid sequences of which revealed high homology with those of hsp60 and an ubiquitin-conjugating enzyme. The nitrilase exhibited maximum activity (91.6 U mg(-1)) at 45 degrees C and pH 8.0. Its preferred substrates, in the descending order, were 4-cyanopyridine, benzonitrile, 1,4-dicyanobenzene, thiophen-2-acetonitrile, 3-chlorobenzonitrile, 3-cyanopyridine, and 4-chlorobenzonitrile. Formation of amides as by-products was most intensive, in the descending order, for 2-cyanopyridine, 4-chlorobenzonitrile, 4-cyanopyridine, and 1,4-dicyanobenzene. The enzyme stability was markedly improved in the presence of D: -sorbitol or xylitol (20% w/v each). p-Hydroxymercuribenzoate and heavy metal ions were the most powerful inhibitors of the enzyme.
authors.affiliation: Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídenská 1083, 14220 Prague, Czech Republic.
authors.forename: Ondrej
authors.initials: O
authors.lastname: Kaplan
authors.forename: Vojtech
authors.initials: V
authors.lastname: Vejvoda
authors.forename: Ondrej
authors.initials: O
authors.lastname: Plíhal
authors.forename: Petr
authors.initials: P
authors.lastname: Pompach
authors.forename: Daniel
authors.initials: D
authors.lastname: Kavan
authors.forename: Pavla
authors.initials: P
authors.lastname: Bojarová
authors.forename: Karel
authors.initials: K
authors.lastname: Bezouska
authors.forename: Martina
authors.initials: M
authors.lastname: Macková
authors.forename: Maria
authors.initials: M
authors.lastname: Cantarella
authors.forename: Vladimír
authors.initials: V
authors.lastname: Jirků
authors.forename: Vladimír
authors.initials: V
authors.lastname: Kren
authors.forename: Ludmila
authors.initials: L
authors.lastname: Martínková
mesh_terms: D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000737:chemistry* / Q000302:isolation & purification*; D001232:Aspergillus fumigatus / Q000201:enzymology; D001234:Aspergillus niger / Q000201:enzymology*; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000619:Aminohydrolases; C021867:nitrilase