Overproduction of D-hydantoinase and carbamoylase in a soluble form in Escherichia coli

The production of D-hydantoinase and carbamoylase from Agrobacterium radiob acter NRRL B11291 using T7 and trc promoters, respectively, was found to ca use protein aggregates in Escherichia coli. We initiated a systematic study aimed at overproducting these two proteins in a soluble form. As a result, the protein aggregate from carbamoylase overproduction could be alleviated with the aid of GroEL/GroES. In contrast, the production of a high level o f D-hydantoinase in an active form can be achieved at low temperature (25 d egrees C) or by the coproduction of DnaJ/DnaK. Overall, with such approache s both recombinant proteins gain more than a four-fold increase in enzyme a ctivity. In addition, by fusion with thioredoxin, D-hydantoinase activity c an be increased 25% more than the unfused counterpart in the presence of Dn aJ/DnaK. These results indicate the success of our approaches to overproduc ing D-hydantoinase and carbamoylase in a soluble form in E. coli.