A high-throughput amenable colorimetric assay for enantioselective screening of nitrilase-producing microorganisms using pH sensitive indicators.
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.
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India. ucbanerjee@niper.ac.in
Anirban
A
Banerjee
Praveen
P
Kaul
Rohit
R
Sharma
U C
UC
Banerjee
D000097:Acetonitriles / Q000032:analysis / Q000378:metabolism; D042481:Alcaligenes faecalis / Q000235:genetics / Q000378:metabolism*; D000619:Aminohydrolases / Q000378:metabolism*; D002021:Buffers; D003124:Colorimetry / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D015203:Reproducibility of Results
D000097:Acetonitriles; D002021:Buffers; C045516:mandelonitrile; D000619:Aminohydrolases; C021867:nitrilase