( colonies wereinvestigated. From the...)Using XL1-Red Mutator Strain to Generate Esterase Variants

Biotransformation

The esterases (wild type and selected mutants) were subjected to the preparative hydrolysis of substrate 1 in phosphate buffer (50 mM, pH 7.5) at 40C. As expected, no reaction was observed either with the wild type (PFE) or in the absence of enzyme. In contrast, by using variant A209D/L181V, stereoselective hydrolysis of substrate 1 occurred, resulting in what is 25% enantiometric excess (%ee) (determined by gas chromatography using a chiral column) for the remaining ester. The stereoselectivity was further confirmed by determining the optical rotation of the substrate and the reaction product. The remaining ester substrate gave an [a]_D20 of + 0.97 , and the produced acid, as expected, rendered a contrary sense of rotation of [a]_D20= - 11.01 after a 10-day reaction time. Mutants derived from the second mutation cycle exhibited less stereoselectivity compared to the variant, A209D/L181V. The low stereoselectivity observed in the esterase-catalyzed resolution of substrate 1 relates to the generally observed weak stereoselectivity of PFE.¹⁰

Conclusions

The results clearly demonstrate that the XL1-Red mutator strain is efficient as an alternative method to direct the evolution of enzymes. Besides being easy to use, XL1-Reds mutation rate compares well to error-prone PCR, while excluding the problems associated with PCR ligation. Because the assay does not require special substrates, such as chromophoric esters, a large number of clones can be quickly identified by their surrounding red color. Additionally, only a few mutation cycles are necessary to identify an esterase variant, which hydrolyzes a sterically-hindered substrate. We expect that direct
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