In situ monitoring of turbid immobilized lipase-catalyzed esterification of oleic acid using fiber-optic Raman spectroscopy

Raman spectroscopy with a fiber-optic probe was used to monitor the immobilized Candida antarctica lipase B (Novozym 435)-catalyzed esterification of oleic acid with ethanol in iso-octane as an organic solvent. The effects of reaction temperature and molar ratio of substrates were studied. An optimal reaction rate was found at 60 °C, beyond which deactivation due to denaturing of the lipase was observed. The variation in molar ratio of substrates suggests that the esterification of oleic acid and ethanol proceeds via a Ping-Pong Bi-Bi mechanism with ethanol exhibiting an inhibitory effect. A good fit was obtained between the experimental results and the best-fit Ping-Pong Bi-Bi mechanism. This current work shows that fiber-optic Raman spectroscopy is indeed a suitable instrument to monitor immobilized lipase-catalyzed reaction in turbid organic systems in situ. Since this approach is reliable, simple to use, allows automatic data acquisition, and accurate, it should be applicable to the detailed kinetic analysis on other immobilized enzymatic reactions as well.