Immobilization of Invertase on Glyoxyl, Amine and Glutaraldehyde Supports: Determination of the Best Derivative for Sucrose Hydrolysis
Goulart, A.J.1; Tavano, O.L.1; Marques, D.P.2; Giordano, R.L.C.3; Monti, R.2
1 Departamento de Bioquímica e Química Tecnológica, IQ–UNESP, Araraquara – SP; 2Departamento Alimentos e Nutrição, FCF–UNESP, Araraquara – SP; 3Departamento de Engenharia Química, UFSCar, São Carlos – SP. e-mail: montiru@fcfar.unesp.br.
Enzymes are biological catalysts that aren't consumed during the process they act. Thus, the yield could be larger if they are immobilized on inert supports and reused. The objective of this work was to determine the best derivative obtained using a commercial invertase immobilized by covalent binding or adsorption on agarose supports. Agarose was activated with 1.7N NaOH, sodium borohydride and glycidol (2,3-epoxy-propanol), and was oxidized to glyoxyl-agarose with 0.1M sodium periodate solution. Amine-agarose was prepared adding 2M ethylenediamine and sodium borohydride; glutaraldehyde-agarose was prepared suspending amine-agarose in 15% glutaraldehyde-0.2M phosphate buffer pH 7.0. Immobilization was achieved suspending the supports in an enzyme solution under mild stirring at 22ºC. Enzyme optima hydrolysis temperature and pH were 40ºC and 5.0, respectively, and experiments to determine thermal stability showed residual activity at about 70% after 24h at 40ºC, pH 5.0. Glyoxyl derivative showed residual activity at about 80%, and amine and glutaraldehyde derivatives retained 100% of activity. Enzyme and derivatives were incubated in 0.05M phosphate buffer pH 7.0, 40ºC, but only glutaraldehyde derivative kept stable at this pH after 24h. The derivatives optima hydrolysis temperatures were 60ºC (glyoxyl), 55ºC (amine) and 65ºC (glutaraldehyde). Soluble enzyme and glyoxyl derivative didn't show product inhibition even when the substract concentration was below 0.012 mmol/mL in the presence of 200 mmol fructose, but the derivatives amine and glutaraldehyde were inhibited in higher or lower degree in all substract concentrations tested. These results allow concluding that the best derivative tested in this work was glyoxyl-agarose-invertase because it presented relatively high optimum hydrolysis temperature, high activity even after 24h at 40ºC, and for didn't present product inhibition even when the inhibitor was at very high concentrations.
Acknowledgements: This work was supported by CNPq, CAPES and Programa de Apoio ao Desenvolvimento Científico (PADC) – Faculdade de Ciências Farmacêuticas – UNESP.
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