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A High-Throughput Amenable Colorimetric Assay for Enantioselective Screening of Nitrilase-Producing Microorganisms Using pH Sensitive Indicators

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India

Praveen Kaul

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India

Rohit Sharma

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India

U. C. Banerjee

Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India, ucbanerjee{at}niper.ac.in

Based on the color change of an indicator due to the release of hydrogen ion from a nitrilase-catalyzed reaction, a rapid colorimetric method was established for the enantioselective screening of nitrilase-producing microorganisms. The formation of acids due to the nitrilase-mediated hydrolysis of nitriles causes a drop in the pH, which in turn results in a change of color of the solution (containing indicator) that can be observed visually. The buffer (0.01 M phosphate, pH 7.2) and indicator (Bromothymol blue, 0.01%) were selected in such a way that both have the same affinity for the released protons. The enantioselectivity of nitrilases was estimated by comparing the hydrolysis of (R)-mandelonitrile with that of racemate under the same conditions. The method was used to screen a library of nitrilase-producing microorganisms, isolated in the authors' laboratory for their ability to enantioselectively hydrolyze mandelonitrile to mandelic acid, an important chiral building block. (Journal of Biomolecular Screening 2003:559-565).

Key Words: nitrilase • indicator • pKa • enantioselectivity • mandelonitrile • mandelic acid • Alcaligenes faecalis

Journal of Biomolecular Screening, Vol. 8, No. 5, 559-565 (2003)
DOI: 10.1177/1087057103256910


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