Immobilization of Carboxypeptidase A into Modified Chitosan Matrixes by Covalent Attachment

Carboxypeptidase A (CPA) is a metalloexopeptidase that catalyzes the hydrolysis of the peptide bonds that are adjacent to the C-terminal end of a polypeptide chain. The enzyme preferentially cleaves over C-terminal L-amino acids with aromatic or branched side chains. This is of main importance for food industry because it can be employed for manufacturing functional foods from different protein sources with reduced hydrophobic amino acid content for patients with deficiencies in the absorption or digestion of the corresponding amino acids. In that way, strategies for effective multipoint covalent immobilization of CPA metalloenzyme on chitosan beads have been developed. The study of the ability to produce several chemical modifications on chitosan molecules before, during and after its coagulation to form carrier beads lead in a protective effect of the polymer matrix. The chemical modification of chitosan through the use of an N-alkylation strategy produced the best derivatives. N-alkyl chitosan derivative beads with D-fructose presented values of 0.86 for immobilization yield, 314.6 IU g(-1) bead for initial activity of biocatalyst and were 5675.64-fold more stable than the free enzyme at 55 A degrees C. Results have shown that these derivatives would present a potential technological application in hydrolytic processes due to both their physical properties, such as low swelling capacity, reduced metal chelation ability and bulk mesoporosity, and increased operational stability when compared with soluble enzyme.