Free and Immobilized Lecitase™ Ultra as the Biocatalyst in the Kinetic Resolution of (E)-4-Arylbut-3-en-2-yl Esters

The influence of buffer type, co-solvent type, and acyl chain length was investigated for the enantioselective hydrolysis of racemic 4-arylbut-3-en-2-yl esters using Lecitase™ Ultra (LU). Immobilized preparations of the Lecitase™ Ultra enzyme had significantly higher activity and enantioselectivity than the free enzyme, particularly for 4-phenylbut-3-en-2-yl butyrate as the substrate. Moreover, the kinetic resolution with the immobilized enzyme was achieved in a much shorter time (24–48 h). Lecitase™ Ultra, immobilized on cyanogen bromide-activated agarose, was particularly effective, producing, after 24 h of reaction time in phosphate buffer (pH 7.2) with acetone as co-solvent, both (R)-alcohols and unreacted (S)-esters with good to excellent enantiomeric excesses (ee 90–99%). These conditions and enzyme were also suitable for the kinetic separation of racemic (E)-4-phenylbut-3-en-2-yl butyrate analogs containing methyl substituents on the benzene ring (4b,4c), but they did not show any enantioselectivity toward (E)-4-(4’-methoxyphenyl)but-3-en-2-yl butyrate (4d).

Enantioselective Hydrolysis of Styrene Oxide and Benzyl Glycidyl Ether by a Variant of Epoxide Hydrolase from Agromyces mediolanus

Enantiopure epoxides are versatile synthetic intermediates for producing optically active pharmaceuticals. In an effort to provide more options for the preparation of enantiopure epoxides, a variant of the epoxide hydrolase (vEH-Am) gene from a marine microorganism Agromyces mediolanus was synthesized and expressed in Escherichia coli. Recombiant vEH-Am displayed a molecular weight of 43 kDa and showed high stability with a half-life of 51.1 h at 30 °C. The purified vEH-Am exhibited high enantioselectivity towards styrene oxide (SO) and benzyl glycidyl ether (BGE). The vEH-Am preferentially converted (S)-SO, leaving (R)-SO with the enantiomeric excess (ee) >99%. However, (R)-BGE was preferentially hydrolyzed by vEH-Am, resulting in (S)-BGE with >99% ee. To investigate the origin of regioselectivity, the interactions between vEH-Am and enantiomers of SO and BGE were analyzed by molecular docking simulation. In addition, it was observed that the yields of (R)-SO and (S)-BGE decreased with the increase of substrate concentrations. The yield of (R)-SO was significantly increased by adding 2% (v/v) Tween-20 or intermittent supplementation of the substrate. To our knowledge, vEH-Am displayed the highest enantioselectivity for the kinetic resolution of racemic BGE among the known EHs, suggesting promising applications of vEH-Am in the preparation of optically active BGE.

Overcoming Water Insolubility in Flow: Enantioselective Hydrolysis of Naproxen Ester

Hydrolytic enantioselective cleavage of different racemic non-steroidal anti-inflammatory drugs (NSAIDs) ester derivatives has been studied. An engineered esterase form Bacillus subtilis (BS2m) significantly outperformed homologous enzymes from Halomonas elongata (HeE) and Bacillus coagulants (BCE) in the enantioselective hydrolysis of naproxen esters. Structural analysis of the three active sites highlighted key differences which explained the substrate preference. Immobilization of a chimeric BS2m-T4 lysozyme fusion (BS2mT4L1) was improved by resin screening achieving twice the recovered activity (22.1 ± 5 U/g) with respect to what had been previously reported, and was utilized in a packed bed reactor. Continuous hydrolysis of α-methyl benzene acetic acid butyl ester as a model substrate was easily achieved, albeit at low concentration (1 mM). However, the high degree of insolubility of the naproxen butyl ester resulted in a slurry which could not be efficiently bioconverted, despite the addition of co-solvents and lower substrate concentration (1 mM). Addition of Triton® X-100 to the substrate mix yielded 24% molar conversion and 80% e.e. at a 5 mM scale with 5 min residence time and sufficient retention of catalytic efficiency after 6 h of use.