Visualization of enzyme-catalyzed reactions using pH indicators: rapid screening of hydrolase libraries and estimation of the enantioselectivity.
The use of pH indicators to monitor hydrolase-catalyzed reactions is described. The formation of acid following an enzyme-mediated hydrolysis causes a drop in the pH that can be visualized by a change in the color of the indicator-containing solution. The best indicators are those showing a color transition within the operational pH range of the hydrolases, like bromothymol blue and phenol red. The enantioselectivity of lipases and esterases can be estimated using single isomers under the same conditions and comparing the color turnover for each one. The method has been tested to quickly evaluate the enantioselectivity of a lipase towards a set of ester substrates and applied to the hierarchical screening of a library of thermophilic esterases.
Morís-Varas
F
F
ThermoGen, Inc., Chicago, IL 60612, USA.
Shah
A
A
Aikens
J
J
Nadkarni
N P
NP
Rozzell
J D
JD
Demirjian
D C
DC
D000097:Acetonitriles / Q000737:chemistry / Q000378:metabolism; D001979:Bromthymol Blue / Q000737:chemistry; D016956:Burkholderia cepacia / Q000201:enzymology; D002384:Catalysis; D002625:Chemistry, Organic / Q000295:instrumentation / Q000379:methods*; D003124:Colorimetry / Q000379:methods; D004798:Enzymes / Q000737:chemistry / Q000378:metabolism*; D004950:Esterases / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000737:chemistry* / Q000378:metabolism; D007202:Indicators and Reagents / Q000737:chemistry*; D019151:Peptide Library; D010637:Phenolsulfonphthalein / Q000737:chemistry; D010710:Phosphates / Q000737:chemistry; D017680:Potassium Compounds / Q000737:chemistry; D012680:Sensitivity and Specificity; D013237:Stereoisomerism
D000097:Acetonitriles; D004798:Enzymes; D007202:Indicators and Reagents; D019151:Peptide Library; D010710:Phosphates; D017680:Potassium Compounds; C045516:mandelonitrile; C013216:potassium phosphate; D006867:Hydrolases; D004950:Esterases; D010637:Phenolsulfonphthalein; D001979:Bromthymol Blue