Medline/PubMed Citation | Related Articles in PubMed | Download to Citation Manager

Biotechnology and Applied Biochemistry (2003) 37, (289–293) (Printed in Great Britain)

PDF

Legacy HTML

A rapid and sensitive fluorometric assay method for the determination of nitrilase activity

Anirban Banerjee, Rohit Sharma and Uttam Chand Banerjee¹

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, Mohali, Punjab-160062, India

Key words: amidase, Berthelot method, fluorescence intensity, nitrilase, nitrile hydratase.

¹To whom correspondence should be addressed (e-mail ucbanerjee@niper.ac.in).

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 NH₃ 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.

Received 31 October 2002/19 December 2002; accepted 28 January 2003

Published as Immediate Publication 28 January 2003, DOI 10.1042/BA20020106

Portland Press Ltd ©2003