Preparation, characterization and application of immobilized carboxypeptidase A

Bovine carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 3.4.17.1) wa s covalently attached to polyacrylamide beads and to silica-based supports, and also adsorbed on polyethylene terephthalate. The highest immobilized a ctivity (125 U g(-1) solid) was achieved when a polyacrylamide bead support (Akrilex C) possessing carboxylic functional groups activated by a water-s oluble carbodiimide was used. The catalytic properties and stability of Akr ilex C-carboxypeptidase A were studied and compared with the corresponding properties of the soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase A was practically identical to that fo r the soluble enzyme (pH 7.5-8.0). The apparent optimum temperature of the immobilized carboxypeptidase A was about 20 degrees C higher than that of t he soluble enzyme. With hippuryl-L-phenylalanine as substrate, K-m app for the immobilized enzyme (1.65 mM) was somewhat higher than K-m for the solub le enzyme (1.07 mM). The conformational stability of the enzyme was markedl y enhanced by the strongly hydrophilic microenvironment. The immobilized ca rboxypeptidase A was used for the C-terminal amino acid analysis of peptide s. (C) 1999 Elsevier Science Inc. All rights reserved.