Strategic selection of hyperthermophilic esterases for resolution of 2-arylpropionic esters.
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.
Sehgal
Amitabh C
AC
North Carolina State University, Department of Chemical Engineering, Raleigh, North Carolina 27695-7905, USA.
Kelly
Robert M
RM
D000595:Amino Acid Sequence; D002175:Candida / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004795:Enzyme Stability; D004950:Esterases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D004952:Esters; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009288:Naproxen / Q000031:analogs & derivatives / Q000737:chemistry*; D015202:Protein Engineering / Q000379:methods*; D020027:Pyrococcus furiosus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016415:Sequence Alignment / Q000379:methods; D020539:Sequence Analysis, Protein / Q000379:methods*; D013045:Species Specificity; D013237:Stereoisomerism; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics
D004952:Esters; D011994:Recombinant Proteins; C487872:naproxen methyl esther; D009288:Naproxen; D004950:Esterases