Purification and characterization of a nitrilase from Aspergillus niger K10 |
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Authors: | Ond?ej Kaplan Vojtěch Vejvoda Ond?ej Plíhal Petr Pompach Daniel Kavan Pavla Bojarová Karel Bezou?ka Martina Macková Maria Cantarella Vladimír Jirk? Vladimír K?en Ludmila Martínková |
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Institution: | (1) Institute of Microbiology, Centre of Biocatalysis and Biotransformation, Academy of Sciences of the Czech Republic, Vídeňská 1083, 14220 Prague, Czech Republic;(2) Department of Biochemistry, Faculty of Science, Charles University Prague, Hlavova 8, 12840 Prague, Czech Republic;(3) Faculty of Food and Biochemical Technology, Institute of Chemical Technology Prague, Technická 5, 16628 Prague, Czech Republic;(4) Department of Chemistry, Chemical Engineering and Materials, University of L’Aquila, Monteluco di Roio, 67040 L’Aquila, Italy |
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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°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. |
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Keywords: | Nitrilase Aspergillus niger Enzymatic nitrile hydrolysis |
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