Production of thermotolerant N-carbamyl-D-amino acid amidohydrolase by recombinant Escherichia coli

A plasmid, pNT4553, was constructed for high level production of N-carbamyl -D-amino acid amidohydrolase (DCase), the thermostability of which has been improved by amino acid substitution. The DCase activity and the stability of the plasmid in the host cells were dependent on the Escherichia coli str ains used. E. coli HB101 was the most suitable host strain among the 13 typ es of E. coli tested. E. coli HB101 exhibited the highest activity, i.e. 6. 36 units/ml of culture broth in 2YT medium (1.6% tryptone, 1.0% yeast extra ct, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultiv ation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, pept one, and protein extract (a mixture of hydrolyzates of corn gluten, wheat g luten and soybean), were found to be suitable as natural nitrogen sources f or both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, a nd protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 mi of medium in a 500ml shaking hash), in which the aeration lev el was estimated to be high, growth and activity reached OD550= 63.8 (17.1m g of dry cell weight/ml of culture broth) and 22.9 units/ml of culture brot h, respectively. The economical hyperproduction of DCase using only inexpen sive constituents for the medium was achieved.