7.2 Screening Methods for Enzymes

Abstract

Analytical methods to measure catalytic activity are critical tools to enable the discovery, purification, optimization, and use of any enzyme. Screening consists of individually testing each sample of a series, such as various enzyme mutants or reaction conditions, for the targeted reaction. Screening can be performed: (1) in living cells expressing the enzyme of interest using chromogenic substrates and indicators on agar plates or fluorescence-activated cell sorting; (2) in microtiter plates using chromogenic and fluorogenic substrates, Förster or fluorescence resonance energy transfer substrates, or indicators of reaction progress; (3) using multiple substrates to address both activity and selectivity simultaneously; (4) with the help of analytical chemistry instrumentation such as high pressure liquid chromatography, gas chromatography, mass spectrometry, and nuclear magnetic resonance- or infrared-spectrometers. Screening performed under conditions relevant for the targeted process and with the appropriate positive and negative controls and with reconfirmation testing for positive hits provides a valuable support to biocatalyst discovery and optimization.