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 strains used. E. coli HB101 was the most suitable host strain among the 13 types 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 extract, and 0.5% NaCl, pH 7.0), and the plasmid was stably maintained by cultivation in 5 types of E. coli including HB101. Casamino acids, NZ-amine, peptone, and protein extract (a mixture of hydrolyzates of corn gluten, wheat gluten and soybean), were found to be suitable as natural nitrogen sources for both enzyme activity and growth. When cultivation was carried out in the presence of high concentrations of glycerol (6.5%) as the carbon source, and protein extract (3.0%) as the nitrogen source, in a small volume of the medium (20 ml of medium in a 500-ml shaking flask), in which the aeration level was estimated to be high, growth and activity reached OD550=63.8 (17.1 mg of dry cell weight/ml of culture broth) and 22.9 units/ml of culture broth, respectively. The economical hyperproduction of DCase using only inexpensive constituents for the medium was achieved.