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

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 (%ee(p) >/= 90) and was thus further investigated. Changes in pH or reaction time showed little improvement in %ee(p) 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.

Thermostable Esterase from a Thermoacidophilic Archaeon: Purification and Characterization for Enzymatic Resolution of a Chiral Compound

“…The carboxylesterase from S. solfataricus P1 (Sso Est1) showed a specific reaction toward the Snaproxen ester in co-solvent reaction conditions. 25) But Est3 displayed a different catalytic pattern toward anarylpropionic methylester substrate, ketoprofen methylester, containing the same chiral center. The selectivity toward substrates observed in previous literature might be elucidated by the fact that the change in the sidechain structure of the substrate leads to a drastic change in reaction rate and enantioselectivity.…”

Carboxylic ester hydrolases from hyperthermophiles

“…The enzyme hydrolyzed the ( R )-ester of racemic ketoprofen methylester and showed an enantiomeric excess of 80% with a conversion rate of 20% in 32 h. In another study, the esterase Sso-Est1 from S. solfataricus P1 (Sehgal et al 2001 ) was identified as homolog to the mesophilic Bacillus subtilis ThaiI-8 esterase (CNP) (Quax and Broekhuizen 1994 ) and Candida rugosa lipase (CRL) (Lee et al 2001 ), which are used for the chiral separation of racemic mixtures of 2-arylpropionic methyl esters. The enzyme was characterized biochemically for its ability to resolve mixtures of ( R,S )-naproxen methyl ester under a variety of reaction environments (Sehgal and Kelly 2002 , 2003 ). Sso-Est1 showed a specific reaction toward the ( S )-naproxen ester in co-solvent reaction conditions with an enantiomeric excess of ≥90%.…”

Purification and Characterization of a Thermostableβ-Mannanase fromBacillus subtilisBE-91: Potential Application in Inflammatory Diseases

“…β -mannanases are widely applied in pulp and paper processing [4], feed [5], food [6], pharmaceutical [7], oil, and textile industries [8] to randomly hydrolyze the β -1,4 mannopyranoside linkage in mannan, galactomannan, glucomannan, and galactoglucomannan.…”