Purification and characterization of nitrilase from Fusarium solani IMI196840

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Authors

VEJVODA Vojtech KUBAC David DAVIDOVA Alzbeta KAPLAN Ondrej SULC Miroslav SVEDA Ondrej CHALOUPKOVÁ Radka MARTINKOVA Ludmila

Year of publication 2010
Type Article in Periodical
Magazine / Source Process Biochemistry
MU Faculty or unit

Faculty of Science

Citation
Web https://doi.org/10.1016/j.procbio.2010.03.033
Doi http://dx.doi.org/10.1016/j.procbio.2010.03.033
Keywords Fusarium solani; Nitrilase; Purification; Characterization; Nitriles
Description Nitrilase activity in Fusarium solani IMI196840 (approx. 1500 Ul(-1) of culture broth) was induced by 2-cyanopyridine. The enzyme was purified by a factor of 20.3 at a yield of 26.9%. According to gel filtration, the holoenzyme was an approx. 550-kDa homooligomer consisting of subunits with a molecular weight of approximately 40 kDa, as determined by SDS-PAGE. Mass spectrometry analysis of the tryptic fragments suggested a high similarity of this enzyme to the hypothetical CN hydrolases from Aspergillus oryzae, Gibberella zeae, Gibberella moniliformis and Nectria haematococca. Circular dichroism and fluorescence spectra indicated that secondary structure content and overall tertiary structure, respectively, were almost identical in nitrilases from F. solani IMI196840 and F. solani O1. The melting temperatures of the enzymes were 49.3 degrees C and 47.8 degrees C, respectively. The best substrates for the purified nitrilase from F. solani IMI196840 were benzonitrile and 4-cyanopyridine, which were hydrolyzed at the rates of 144 and 312 U mg(-1) protein, respectively, under the optimum conditions of pH 8 and 45 C. The enzyme was highly chemoselective, producing <= 2% amides as by-products.
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