No 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.

No abstract

The enantiomeric resolution of 2-aryl propionic esters by hyperthermophilic and mesophilic esterases was found to be governed by contrasting thermodynamic mechanisms. Entropic contributions predominated for mesophilic esterases from Candida rugosa and Rhizomucor miehei, while enthalpic forces controlled this resolution by the esterase from the extremely thermoacidophilic archaeon, Sulfolobus solfataricus P1. This disparity in thermodynamic mechanism can be attributed to the differences in conformational flexibility of mesophilic and thermophilic enzymes as they relate to the temperature range (4-70 degrees C) examined.

The interactive effects of temperature and cosolvents on the kinetic and structural features of a carboxylesterase from the extremely thermoacidophilic archaeon Sulfolobus solfataricus P1 (Sso EST1) were examined. While dimethylformamide, acetonitrile, and dioxane were all found to be deleterious to enzyme function, dimethyl sulfoxide (DMSO) activated Sso EST1 to various extents. This was particularly true at 3.5% (v/v) DMSO, where k(cat) was 20-30% higher than at 1.2% DMSO, over the temperature range of 50-85 degrees C. DMSO compensated for thermal activation in some cases; for example, k(cat) at 60 degrees C in 3.5% DMSO was comparable to k(cat) at 85 degrees C in 1.2% DMSO. The relationship between DMSO activation and enzyme structural characteristics was also investigated. Nuclear magnetic resonance spectroscopy and circular dichroism showed no gross change in enzyme conformation with 3.5% DMSO between 50 and 80 degrees C. However, low levels of DMSO were shown to have a small yet significant change in enzyme conformation. This was evident through the reduction of Sso EST1's melting temperature and changes in the microenvironment of the enzyme's tyrosine and tryptophan residues at 3.5% versus 1.2% (v/v) solvent. Finally, activation parameter analysis based on kinetic data, at 1.2% and 3.5% DMSO, implied an increase in conformational flexibility with additional cosolvent. These results suggest the activating effect of DMSO was related to small changes in the enzyme's structure resulting in an increase in its conformational flexibility. Thus, in addition to their use for solubilizing hydrophobic substrates in water, cosolvents may also serve as activators in applications involving thermostable biocatalysts at sub-optimal temperatures.