Simultaneous purification and immobilization of Candida rugosalipase on superparamagnetic Fe3O4nanoparticles for catalyzing transesterification reactions

Abstract

The Fe₃O₄ superparamagnetic nanoparticles (about 5 nm in size) were coated with a surfactant Tween 80, fatty acids and polyethyleneimine (PEI). The best results were obtained with PEI coated nanoparticles. The coated particles selectively adsorbed Candida rugosalipase from a commercial preparation resulting in about 2 fold purification. The immobilized lipase showed 110 times increase in initial rates of transesterification of ethyl butyrate with butanol in anhydrous hexane over “straight from the bottle” commercial preparation. The opening of the molecular lid over the active site of lipase is likely to be a contributing factor for this enhancement. Spreading of the enzyme over the large surface of the nanoparticles is also expected to reduce mass transfer constraints. A spinning cell accessory fabricated for the commercial CD instrument allowed us to record CD spectra of the lipase immobilized on the nanoparticles. CD spectra showed that no significant changes in the secondary structure took place upon immobilization. The immobilized nanobiocatalyst also was found to be efficient in kinetic resolution of 1-phenylethanol with 99% ee_p and E = 412. The commercial preparation did not show significant conversion and hence no kinetic resolution could be obtained. Hence the nanobiocatalyst design should prove useful in both organic synthesis and kinetic resolutions using enzymes.