Rational Protein Design of Paenibacillus barcinonensis Esterase EstA for Kinetic Resolution of Tertiary Alcohols

Abstract

Protein engineering is a very powerful tool to optimize enzymes for specific applications and thus provide important chiral building blocks such as tertiary alcohols. By use of structural comparisons, esterase from Paenibacillus barcinonensis (EstA) was engineered to convert tertiary alcohol esters with excellent enantioselectivity. Whereas the wild‐type enzyme converts 1,1,1‐trifluoro‐2‐phenylbut‐3‐yn‐2‐yl acetate with very low activity and enantioselectivity (E=12, at 4 °C), several mutants show a significantly increased enantioselectivity, for example E>100 for mutant EstA–AGA, under the same reaction conditions. Furthermore, the range of tertiary alcohols obtained in enantiopure form was also broadened for EstA mutants.

Kinetic resolution of esters from secondary and tertiary benzylic propargylic alcohols by an improved esterase-variant from Bacillus sp. BP-7

“…A widening of the oxyanion hole area, acquired by the amino acid change produced on wild-type EstBP7 could then account for a better accommodation of pyridinesubstituted tertiary alcohols (Scheme 1, substrates 11, 12) with the active site of the enzyme. On the contrary, the moderate-tohigh enantioselective E-values obtained with CF 3 -containing esters (5)(6)(7)(8)(9) suggests that the widening originated in the oxyanion hole would not be enough to accommodate such kind of substrates. Interestingly, drugs used as HIV transcriptase inhibitors such as Efavirenz contain a chiral structure that resembles that of CF 3 -containing esters [35].…”

“…These variants were rationally designed to investigate the influence of the oxyanion hole motif sequence on the activity and especially to analyze their enantioselectivity in the kinetic resolution of a range of tertiary alcohols. This strategy seemed to be suitable for our purpose based on previous successful studies [6,25]. Furthermore, attempts to generate activity towards esters of tertiary alcohols into GX-type esterases using methods of directed evolution still remained unsuccessful [24].…”

“…Lipases and esterases are valuable enzymes to carry out such processes [6,7]. Indeed, they are stable in organic solvents and are able to catalyze a wide range of transformations, including both hydrolysis as well as ester-forming reactions, in a highly chemo-, regio-, and stereoselective manner, necessary for efficient kinetic resolutions [8,9].…”

Lipase-catalyzed remote kinetic resolution of citalopram intermediate by asymmetric alcoholysis and thermodynamic analysis

“…The enantioselectivity of the enzyme-catalyzed resolution of tertiary alcohols was generally low due to the adverse steric hindrance of the substrates [3][4][5][6]. To improve the interpretation of the kinetic resolution of tertiary alcohols, the thermodynamic analysis can be applied as efficient tools.…”

Double substituted variant of Bacillus amyloliquefaciens esterase with enhanced enantioselectivity and high activity towards 1-(3′,4′-methylenedioxyphenyl)ethyl acetate

“…Different strategies such as ep-PCR (Reetz et al 1997;Ma et al 2013;Qin et al 2013), saturation mutagenesis (Reetz et al 2006;Carballeira et al 2007;Kotik et al 2011), and rational design (Ema et al 2005(Ema et al , 2010Heinze et al 2007;Bordes et al 2009;Bassegoda et al 2010) have been successfully applied to generate enzyme variants with excellent enantioselectivity. The paranitrobenzyl esterase containing a GGG(A)X-motif has high catalytic activity towards esters of chiral tertiary alcohols (Henke et al 2003), and great efforts have been made to improve its enantioselectivity for tertiary alcohols by protein engineering, providing insights into the molecular mechanism of enzymes (Heinze et al 2007;Kourist et al 2007;Barbayianni et al 2009;Wiggers et al 2009).…”