A rapid and sensitive fluorometric assay method for the determination of nitrilase activity.
A rapid, simple and sensitive fluorometric assay method for the determination of nitrilase activity is described. 3-Cyanopyridine was hydrolysed to nicotinic acid by Rhodococcus rhodochrous and the liberated NH(3) was allowed to react with buffered o -phthaldialdehyde-2-mercaptoethanol solution (pH 7.4) to form a fluorochrome. The fluorescence intensity was found to be stable after 20 min incubation at room temperature, and the optimum pH for the reaction was found to be 7.4. The fluorescence intensity was linearly related to enzyme activity with the substrate concentration ranging from 100 to 1000 mM. The activity determined by the proposed method correlates ( r =0.9625) well with the established Berthelot method. The proposed method is more sensitive than the existing methods for the determination of nitrilase activity.
Banerjee
Anirban
A
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, Mohali, Punjab-160062, India.
Sharma
Rohit
R
Banerjee
Uttam Chand
UC
D000619:Aminohydrolases / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry*; D000641:Ammonia / Q000737:chemistry* / Q000378:metabolism; D004789:Enzyme Activation; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D005470:Fluorometry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000737:chemistry; D009525:Niacin / Q000096:biosynthesis*; D011725:Pyridines / Q000378:metabolism*; D015203:Reproducibility of Results; D012240:Rhodococcus / Q000378:metabolism*; D012680:Sensitivity and Specificity; D009764:o-Phthalaldehyde / Q000737:chemistry*
D005456:Fluorescent Dyes; D011725:Pyridines; D009525:Niacin; D008623:Mercaptoethanol; D009764:o-Phthalaldehyde; D000641:Ammonia; D000619:Aminohydrolases; C021867:nitrilase; C020729:3-cyanopyridine