IMMOBILIZATION OF N-CARBAMYL-D-AMINO ACID AMIDOHYDROLASE

N-Carbamyl-D-amino acid amidohydrolase (DCase), produced with recombin ant Escherichia coil cells using a cloned gene from Agrobacterium sp. strain KNK712, has been immobilized for use in the production of D-ami no acids. The porous polymers, Duolite A-568 and Chitopearl 3003, were much better than other resins for the activity and stability of the a dsorbed enzyme. The activity of DCase expressed on Duolite A-568 and C hitopearl 3003 amounted to 96 units/g-wet-resin and 91 units/g-wet-res in, respectively. DCase immobilized on Duolite A-568 was found to be m ost stable at about pH 7, and it was further stabilized by reductants such as dithiothreitol, L-cysteine, cysteamine, and sodium hydrosulfit e. The stability during the repeated batch reactions was greatly impro ved when dithiothreitol was in the reaction mixture, and the higher cr osslinking degree with glutaraldehyde also stabilized the immobilized enzyme. After 14 times repeated reactions, the remaining activity of t he immobilized enzyme cross-linked with 0.1% and 0.2% of glutaraldehyd e, and 0.2% of glutaraldehyde with dithiothreitol in the reaction mixt ure was 12%, 18%, and 63%, respectively. DCase produced with Pseudomon as sp. strain KNK003A and Pseudomonas sp. strain KNK505, which are the rmotolerant soil bacteria, and that with Agrobacterium sp. strain KNK7 12 were also immobilized on Duolite A-568. The stability of the enzyme s of thermotolerant bacteria during reactions was superior to that of Agrobacterium sp. strain KNK712, though the activity was lower than th at of strain KNK712.