Bovine carboxypeptidase A (peptidyl-L-amino-acid hydrolase, EC 184.108.40.206) was covalently attached to polyacrylamide beads and to silica-based supports, and also adsorbed on polyethylene terephthalate. The highest immobilized activity (125 U g-1 solid) was achieved when a polyacrylamide bead support (Akrilex C) possessing carboxylic functional groups activated by a water-soluble carbodiimide was used. The catalytic properties and stability of Akrilex 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 for the soluble enzyme (pH 7.5-8.0). The apparent optimum temperature of the immobilized carboxypeptidase A was about 20°C higher than that of the 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 soluble enzyme (1.07 mM). The conformational stability of the enzyme was markedly enhanced by the strongly hydrophilic microenvironment. The immobilized carboxypeptidase A was used for the C-terminal amino acid analysis of peptides. Copyright (C) 1999 Elsevier Science Inc.
- Carboxypeptidase A
- Covalently immobilized
- Immobilized carboxypeptidase A
- Peptide digestion
- Polyacrylamide type
ASJC Scopus subject areas
- Applied Microbiology and Biotechnology