Improving enantioselectivity of lipase from Candida rugosa by carrier-bound and carrier-free immobilization

• Enantioselective reactions were carried out with carrier-bound and carrier-free biocatalysts of lipase from Candida rugosa.
• Lipases-catalyzed enantioselective reactions were significantly enhanced by coupling immobilization techniques and reaction medium conditions.
• The strategies employed herein allowed high enantioselective resolutions of (S)-1-phenylethanol (E > 200) and the ethyl (R)-2-hydroxy-4-phenylbutyrate (E = 39).

The enantioselectivity of carrier-bound and carrier-free immobilized lipase from Candida rugosa (CRL) was studied. CRL was immobilized in six agarose-based carriers functionalized with different reactive groups and in two different CRL cross-linked aggregates. Both, activity and enantioselectivity of all the immobilized lipase preparations were evaluated with different racemic esters under different reaction conditions (temperature, pH and solvent polarity). A strong effect of reaction media and immobilization protocol on enzyme activity and selectivity was found. Enzyme immobilization and reaction engineering allowed us obtaining the best immobilization protocol and reaction conditions to achieve high activity and enantioselectivty of CRL as heterogeneous catalyst. CRL immobilized on an agarose-based carrier activated with primary amino groups preferentially hydrolyzed (S)-phenylethyl acetate with E > 200 under pH 7, 4 °C and 30% of acetonitrile. On the other hand, CRL aggregated and cross-linked through their carboxylic groups preferentially hydrolyzed the (S)-isomer of ethyl 2-hydroxy-4-phenylbutyrate with an E = 39 under pH 5, 4 °C and 30% of acetonitrile. This work demonstrates the success of the combinatorial enzyme engineering for the production of highly enantioselective heterogeneous biocatalysts by screening different immobilization protocols and reaction media conditions.

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