Immobilization of Candida rugosa Lipase on Different Ionic Supports to Improve the Enantioselectivity on Mandelic Acid Resolution

This work describes the improvement in enantioselectivity on the resolution of mandelic acid catalyzed by the Candida rugosa lipase (CRL) immobilized and stabilized on two different ionic supports. CRL was quickly immobilized on anionic diethylaminoethyl (DEAE) support and, due to the low content of positive charges on enzyme surface; the enzyme could only be successfully immobilized on cationic carboxymethyl support after chemical amination. Immobilized the enzyme presented higher thermal stability and higher stability to pH than the free enzyme. In relation to the free-enzyme, the DEAE derivative was 2-fold more stable in acid pH, while the carboxymethyl derivative was 2-fold more stable in alkaline pH. When incubated at pH 7.0 and 50 oC, the carboxymethyl derivative was more stable retaining 80% of activity even after 7 h incubation, and the DEAE derivative presented half-life of 6.6 h. Due to this promising characteristics, both CRL derivatives were evaluated on the hydrolysis of (R,S)-mandelic acid ethyl ester under different pH. The CRL immobilized on DEAE support presented stereochemical preference for the R isomer in pH 5.0 and 7.0, while in pH 9.0 the hydrolysis of the S isomer hydrolysis was faster with a higher E-value of 21.2. On the other hand, the carboxymethyl derivative showed opposite results regarding stereochemical preference with higher E-value at pH 5.0 (E > 200) demonstrating excellent enantioselective transesterification towards the S-isomer of mandelic acid with a theoretical 50% conversion yield and a 99.9% enantiomeric excess.