Directed Evolution - Enzyme Tuning Toward Higher Selectivity

Directed Evolution - Enzyme Tuning Toward Higher Selectivity 

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. 

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. 

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]. 

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. 

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