Immobilization of Candida antarctica Lipase B on Magnetic Poly(Urea-Urethane) Nanoparticles.

Abstract

Poly(urea-urethane) (PUU) nanoparticles with encapsulated superparamagnetic magnetite (Fe3O4) were obtained by interfacial miniemulsion polymerization and used as support for immobilization of lipase B from Candida antarctica (CALB). CALB enzyme was immobilized on magnetic PUU nanoparticles in two steps. The enzyme was immobilized in the lyophilized nanoparticles (magnetic PUU) after the support synthesis in phosphate buffer (pH 7.6) containing CALB, by the contact between nanoparticles and enzymatic solution. The mixture was incubated at 30°C in an orbital shaker during 0.5 until 6h to determine the time for maximum immobilization efficiency. The enzyme activity was determined by esterification reactions between lauric acid and propanol. Residual activities above 95% in relation to free enzyme were obtained in 1h of immobilization with enzyme concentration of 0.55mg/mL. FTIR spectrum and SEM-FEG images were used to confirm the presence of CALB on magnetic support after immobilization and stability of support even after immobilization process, respectively. Thermal (40, 60, and 80°C) and pH (pH 4, 7, and 10) stabilities, storage stability, and reuse were evaluated. CALB immobilized derivatives showed high stabilities with residual activities of 95, 100, and 100% at 40, 60, and 80°C, respectively, in 6h of incubation. After incubation in different pH values, CALB immobilized derivative presented activities of 81, 76, and 69% in relation to activities in the beginning of the stabilization process in pH 4, 7, and 10, respectively. Furthermore, CALB immobilized derivative reduces only 15% of its activity after 30days of storage at 4°C. Reuse results showed that immobilized CALB on magnetic PUU nanoparticles led to 95% of geranyl oleate conversion after 8cycles of application demonstrating high stability of the CALB immobilized derivative under different conditions.