Strategic Selection of Hyperthermophilic Esterases for Resolution of 2‐Arylpropionic Esters

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Abstract
Homologues to Carboxylesterase NP and Candida rugosa lipase, used for the chiral separation of racemic mixtures of 2‐arylpropionic methyl esters, were identified by BLAST searches of available genome sequences for hyperthermophilic microorganisms. Two potential candidates were identified: a putative lysophospholipase from Pyrococcus furiosus (Pfu‐LPL) and a carboxylesterase from Sulfolobus solfataricus P1 (Sso‐EST1). Although both enzymes showed hydrolytic preference toward the (S) methyl ester, only Sso‐EST1 yielded highly optically pure (S) naproxen (%eep ≥ 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %eep or E values with Sso‐EST1. However, the addition of 25% methanol resulted in a 25% increase in E. The effect of various cosolvents on the enantiomeric ratio showed no correlation with the log P or dielectric constant values of the solvent. However, an inverse relationship between E and the denaturation capacity (DC) of the water miscible cosolvents was observed. This was attributed to an increase in enzyme flexibility with increasing solvent DC values leading to a concomitant reduction in the resolving power of Sso‐EST1. The results here show that although bioinformatics tools can be used to select candidate biocatalysts for chiral resolution of 2‐arylpropionic esters, biochemical characterization is needed to definitively determine functional characteristics.

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