Macroporous Beads for Lipase Immobilization

Lipase isolated from Arthrobacter sp. (RRLJ-1, MTCC No. 5125, named ABL), is effective in resolving a wide range of racemic drug intermediates. In this study, ABL was immobilized on a series of synthetic macroporous epoxy copolymers beads with varying pore sizes, surface area and hydrophobicity. Poly(glycidyl methacrylate-co-ethylene dimethacrylate) beads, with 75% crosslink density and 10% of epoxy groups modified with dibutyl amine [GMA-EGDM-75 (10% DBA)] had a pore volume of 0.77 mL/g and a surface area of 86.05 m ² /g; these beads were optimally suitable for ABL immobilization. The covalent binding of the lipase was optimized by varying the ionic strength, buffers, pH, temperature and time. The optimal binding was achieved in 100 mM phosphate buffer at 4°C, pH 7.0 in three hours. Under these conditions the polymer retained 34 units and 12 mg of ABL per gram. Immobilized ABL displayed enhanced thermal, organic solvent and pH stability compared to the free enzyme. The immobilized enzyme was used repeatedly (fifteen cycles) to resolve the fluoxitine intermediate (racemic ethyl-3-hydroxy-3-phenyl propanoate) without any loss in stereospecificity. The resolution time of fluoxitine intermediate was reduced to almost half (from 84 to 48 hours) by using the immobilized enzyme.