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.
Banerjee
Anirban
A
Department of Biotechnology, National Institute of Pharmaceutical Education and Research, Sector-67, S. A. S. Nagar, Punjab 160 062, India. ucbanerjee@niper.ac.in
Kaul
Praveen
P
Sharma
Rohit
R
Banerjee
U C
UC
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