Preparation, characterization, and application of a novel immobilized carboxypeptidase B

Abstract

Pig pancreas carboxypeptidase B has been immobilized by covalent attachment to a polyacrylamide-type bead support possessing carboxylic functional groups activated by water-soluble carbodiimide. The optimum conditions of immobilization were determined. The activation of the support and the coupling reaction were performed in 0.1M sodium citrate/sodium phosphate buffer (pH 4.5) using a support-carbodiimide-enzyme weight ratio 4:8:1 at 0‐4 °C. Under such conditions, the highest activity achieved was 6700 U/g solid. The catalytic properties and stability of immobilized carboxypeptidase B were studied and compared with the corresponding properties of the soluble enzyme. The specific activity of the immobilized enzyme calculated on bound protein basis was about 70% of that of soluble enzyme. The optimum pH for the catalytic activity of the immobilized carboxypeptidase B was practically identical with that of soluble enzyme (pH 7.6‐7.7). The apparent optimum temperature of the immobilized carboxypeptidase B was about 7°C higher than that of the soluble enzyme. With hippuryl-L-arginine as substrate, K_mapp value of the immobilized enzyme was tenfold higher than the K_m value of the soluble enzyme. The conformational stability of the enzyme was markedly enhanced by the strongly hydrophylic microenvironment in a wide temperature and pH range. The immobilized carboxypeptidase B was used for stepwise digestion of cytochrome C.