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 610 nm 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. © 2007 Elsevier Inc. All rights reserved.

Ämnesord

NATURVETENSKAP  -- Kemi -- Organisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Organic Chemistry (hsv//eng)

NATURVETENSKAP  -- Biologi -- Biokemi och molekylärbiologi (hsv//swe)

NATURAL SCIENCES  -- Biological Sciences -- Biochemistry and Molecular Biology (hsv//eng)

Nyckelord

Dehalogenation

Glutathione transferase

Iodide

Iodoalkane

Screening

alkane

glutathione transferase A1

haloolefin

iodine

mutant protein

article

cell lysate

colorimetry

cost

enzyme activity

enzyme analysis

enzyme engineering

enzyme specificity

enzyme substrate

high throughput screening

human

microtiter plate assay

nonhuman

priority journal

protein expression

Enzyme Activation

Microchemistry

Titrimetry

Biochemistry

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