Immobilized Enzymes

I. Immobilization of Carboxypeptidase CN, a Preparation from Citrus natsudaidai HAYATA

抄録

Carboxypeptidase CN (CPase CN^*) [EC 3. 4. 12. 1], a preparation from the exocarp of Citrus natsudaidai HAYATA, was immobilized by conjugating the enzyme to AE-cellulose through glutaraldehyde at various pH values with or without pretreatment with a competitive inhibitor, β-phenylpropionic acid. The inhibitor did not protect the enzyme from some loss of activity on immobilization. The specific activity toward Z-Glu-Phe of the preparation (AEC-CPase CN) obtained by conjugation at pH 6.0 without pretreatment with the inhibitor ranged from 24.0 to 59.6% of the activity of native CPase CN. The relative activities of AEC-CPase CN toward N-substituted dipeptides, expressed as a percentage of the activity toward Z-Glu-Phe, were higher than those of native CPase CN, although the specific activities of the former were lower than those of the latter. The values of K_m, V_max, and apparent activation energy for AEC-CPase CN were lower than those for native CPase CN. AEC-CPase CN could be stored at 4°C in 0.1M citrate buffer, pH 5.5, for five months with virtually no loss of activity. It possessed activities corresponding to 74 and 47% of its original activity after seven successive assays performed at 35 and 50°C, respectively. It removed four amino acid residues sequentially from the C-terminus of oxidized lysozyme [EC 3. 2. 1. 17].