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Directed Evolution - Enzyme Tuning Toward Higher Selectivity

Directed Evolution - Enzyme Tuning Toward Higher Selectivity [Pg.110]

An important breakthrough in HTS ee assays came from the group of Reetz in late 1990, with the introduction of mass spectroscopy (MS)-based procedures [90]. These methods use special asymmetrically isotope-labeled compounds. Enzymatic transformations of these compounds usually lead to two pseudoenantiomeric compounds whose relative concentration can be estimated using MS techniques. [Pg.110]

Products 21 and 22 obtained in this reaction differ in their ESI-MS spectra, and the difference in the abundance of respective signals can be expressed quantitatively. Studies have shown that the pseudo-enantiomeric-excess values obtained in this way are in agreement 5% with the data obtained by chromatographic methods, which is sufficient for studying relative values and choosing most selective mutants. [Pg.111]

Initial approaches to directed evolution of enzymes rested upon the introduction of random mutations in random sites of the enzyme by the use of the error-prone PCR technique [92] or on the DNA-shuffling method [93]. Extensive research has also been reported in which every amino acid site in an enzyme was systematically subjected to saturation mutagenesis [94]. [Pg.111]

Pseudomonas aeruginosa lipase-catalyzed hydrolysis of racemic ester 23 proceeds with very low enantioselectivity E = 1.1). Sequential use of error-prone PCR, saturation mutagenesis at chosen spots and DNA shuffling resulted in the formation of a mutant whose enantioselectivity was over 50. [Pg.111]




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Direct selection

Directed enzymes

Directed evolution

Directional selection

Enzyme evolution

Enzyme selection

Enzyme selectivity

Enzymes tuning

Evolution direction

Evolution selection

Selectivity enzyme tuning

Selectivity tuning

Tuning

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