CONICET | Buscador de Institutos y Recursos Humanos

The aim of this work was to development carboxypeptidase A immobilization protocol into hybrid-chitosan matrixes using different polymers on the production of derivatives by multipoint covalent attachment, as well as the activation influence on the obtained derivatives when compared to the free enzyme. Hybrid-chitosan beads were prepared by dissolving in a solution of acetic acid 5%. The obtained solution was dropped into a gently stirred NaOH 0.1M solution. Afterwards, the formed beads were activated with glutaraldehyde or epichlorohydrin. A solution of carboxypeptidase A in bicarbonate buffer 100mM pH 10.05 was added to the activated support and was kept under mild stirring at 25°C for 3h followed by reduction using NaBH4 for 30 minutes. Enzyme activity was assessed via spectrophotometer analysis using hippuryl-L-phenylalanine 1mM. The product released by the substrate hydrolysis, in 25mM tris-HCl buffer, pH 7.5, containing 500 mM NaCl at 25°C was measured at 254 nm. One unit (UH-Phe) was defined as the amount of enzyme that hydrolyses 1.0 mmol of hippuryl-L-phenylalanine per minute at pH 7.5 and 25°C. The derivatives were analyzed as immobilization yield, coupling yield and stabilization factor at 55°C as well as, assays of carboxypeptidase A maximum loading, pH and temperature dependence activity and determination of kinetic and mass transport parameters using hippuryl-L-phenylalanine as substrate at 37°C. The results showed that immobilization via epichlorohydrin presented 40% (8.76) more stable derivatives than glutaraldehyde ones (5.25), on the other hand showed lower immobilization and coupling yield and both derivatives activated by glutaraldehyde and epichlorohydrin presented diffusion limitations with Deff of 5.68*10-12 and 5.23*10-12 m2.s-1 respectively.

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