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.
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