UV/Vis-based ee assays

Several other UV/Vis-based ee assays have been developed, but their general application in directed evolution remains to be demonstrated 66-70). One of them is a well- designed screening system based on enzyme immunoassays (85). The success of the ee assay depends upon the availability of specific antibodies that are easily raised for almost any chiral product of interest. In a given reaction, two antibodies are needed, one that measures the product concentration and the other that measures the amount of one of the enantiomers. About 1000 ee determinations are possible per day, the precision amounting to + 9% (85). 

The first ee-assay designed to handle a reasonably large number of samples (400-700 ee-determi-nations per day) was a rather crude UV/Vis-based screening system for the lipase-catalyzed... 

The first high-throughput ee assay used in the directed evolution of enantioselective enzymes was based on UV/Vis spectroscopy (16,74). It is a crude but useful screening system that is restricted to the hydrolytic kinetic resolution of racemic / -nitrophenyl esters catalyzed by lipases or esterases. The development of this assay arose from the desire to evolve highly enantioselective mutants of the lipase from Pseudomonas aeruginosa as potential biocatalysts in the hydrolytic kinetic resolution of the chiral ester rac-. The wild type leads to an E value of only 1.1 in slight... 

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