Colorimetric endpoint assay for enzyme-catalyzed iodide ion release for high-throughput screening in microtiter plates.
Efforts are being made to engineer enzymes with enhanced activities against haloalkanes, a toxicologically important class of compounds widely used and frequently occurring in the environment. Here we describe a facile, inexpensive, and robust method for the screening of libraries of mutated enzymes with iodoalkane substrates. Iodide formed in the enzymatic reaction is oxidized to iodine, which in the presence of starch gives blue color that can be measured at 610nm or scored with the human eye. The assay can be performed with enzymes in crude cell lysates in 96-wells microtiter plates. Expression clones of several glutathione transferases showed diverse activities with different iodoalkanes, and a mutant library of human glutathione transferase A1-1 expressed variants with enhanced substrate selectivities.
Kurtovic
Sanela
S
Department of Biochemistry and Organic Chemistry, Uppsala University, BMC, Box 576, SE-75123 Uppsala, Sweden.
Jansson
Ronnie
R
Mannervik
Bengt
B
D003124:Colorimetry / Q000379:methods*; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000737:chemistry*; D007455:Iodine / Q000737:chemistry*; D008832:Microchemistry / Q000295:instrumentation* / Q000379:methods*; D017186:Titrimetry / Q000295:instrumentation* / Q000379:methods*
D007455:Iodine; D005982:Glutathione Transferase