Evolution of enantioselectivity: selection of improved hydrolase variants

The introduction of enantiopure active substances is enforced through stricter regulations of the US FDA, enantiopurity is required. Thus, cost-effective processes for the synthesis of enantiopure (precursors of) pharmaceutical compounds are needed. In time, the value of biocatalysis in industrial syntheses has increased. To improve the properties of nature’s catalysts, directed evolution has emerged as a powerful tool to make enzymes more suitable as industrial biocatalysts.
The focus was on the use of lipase A (LipA) of Bacillus subtilis 168 for the production of enantiopure 1,2-O-isopropylidene-sn-glycerol (IPG), a precursor in the synthesis of beta-blockers. As the enantioselectivity of the wild type enzyme is directed towards the unwanted enantiomer R-(-)-IPG, both directed evolution and rational design were applied to invert the enzyme’s enantioselectivity.
Saturation mutagenesis was performed on regions near the active site of LipA. Variants from these libraries were selected using phage display and growth selection. Mutants with an inverted and improved enantioselectivity towards the desired enantiomer S-(+)-IPG were selected and characterised.
As LipA does not have a lid structure controlling access to the active site, insertion of a lid-like structure in LipA could improve enantioselectivity. So-called loop mutants were rationally designed by introducing longer loops from homologous enzymes to extend a loop lining the active site cleft in LipA. The constructed loop mutants proved to be active, having an inverted enantioselectivity towards the wanted enantiomer S-(+)-IPG.
This work provides novel perspectives in the evolution of enzyme enantioselectivity, and can find an application in the improved synthesis of enantiopure beta-blockers.

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