RESUMO
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 6 h 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 1 h of immobilization with enzyme concentration of 0.55 mg/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 6 h 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 30 days of storage at 4 °C. Reuse results showed that immobilized CALB on magnetic PUU nanoparticles led to 95 % of geranyl oleate conversion after 8 cycles of application demonstrating high stability of the CALB immobilized derivative under different conditions.
Assuntos
Candida/enzimologia , Enzimas Imobilizadas/metabolismo , Proteínas Fúngicas/metabolismo , Lipase/metabolismo , Nanopartículas de Magnetita/química , Poliuretanos/farmacologia , Ureia/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Ésteres/metabolismo , Concentração de Íons de Hidrogênio , Nanopartículas de Magnetita/ultraestrutura , Microscopia de Fluorescência , Tamanho da Partícula , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
With the aim of studying the best method for the interaction of polyurethane (PU) foam and Candida antarctica lipase B, different methods of CalB immobilization were studied: adsorption (PU-ADS), bond (using polyethyleneimine) (PU-PEI), ionic adsorption by PEI with cross-linking with glutaraldehyde (PU-PEI-GA) and entrapment (PU). The characterization of immobilized enzyme derivatives was performed by apparent density and Fourier transform infrared spectroscopy. The free enzyme and enzyme preparations were evaluated at different pH values and temperatures. The highest enzyme activity was obtained using the PU method (5.52 U/g). The methods that stood out to compare the stabilities and kinetic parameters were the PU and PU-ADS. Conversions of 83.5 and 95.9 % for PU and PU-ADS derivatives were obtained, in 24 h reaction, using citronella oil and propionic acid as substrates.