Development of Carboxipeptidase A immobilization protocol into hybrid-chitosan matrices by multipoint covalent attachment

ADRIANO, WELLINGTON; MAMMARELLA, ENRIQUE; RUBIOLO, AMELIA; GIORDANO, RAQUEL

Curitiba, Brasil

Simposio; XVI Simpósio Nacional de Bioprocessos – Sinaferm 2007; 2007

Universidade Federal de Paraná

Enzymes are ideal biocatalysts to produce and transform bioactive compounds. The transformations of proteins in high value food, as protein hydrolysates with low contents of phenylalanine are an important issue for chemical and pharmaceutical industries. Moreover, protein hydrolysates free of phenylalanine could be ingested by patients with phenylketonuria, preventing the outcome of brain injures. It is possible to produce an enzymatic hydrolysate with low contents of aromatic amino acids by sequential hydrolysis of proteins. The proposed enzymatic route to obtain this product includes hydrolysis using carboxypeptidase A. However, enzymes are soluble, fragile and expensive and their use for industrial processes requires immobilization and stabilization. Thus, 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 25ºC was measured at 254 nm. One unit (UH-Phe) was defined as the amount of enzyme that hydrolyses 1.0 μmol 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. The preliminary result showed that immobilization via epichlorohydrin presented 40% more stable derivatives than glutaraldehyde ones, on the other hand showed lower immobilization and coupling yield