Preparation, characterization, and application of a novel immobilized carboxypeptidase B.
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 0.1 M sodium citrate/sodium phosphate buffer (pH 4.5) using a support-carbodiimide-enzyme weight ratio 4:8:1 at 0-4 degrees 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 degrees C higher than that of the soluble enzyme. With hippuryl-L-arginine as substrate, Kmapp value of the immobilized enzyme was tenfold higher than the Km 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.
Südi
P
P
Reanal Factory of Laboratory Chemicals, Budapest, Hungary.
Dala
E
E
Szajáni
B
B
D000818:Animals; D043424:Carboxypeptidase B; D002268:Carboxypeptidases / Q000378:metabolism*; D002384:Catalysis; D003574:Cytochrome c Group / Q000378:metabolism; D004355:Drug Stability; D004800:Enzymes, Immobilized / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D010179:Pancreas / Q000201:enzymology; D013552:Swine; D013696:Temperature
D003574:Cytochrome c Group; D004800:Enzymes, Immobilized; D002268:Carboxypeptidases; D043424:Carboxypeptidase B