Production of D−Amino Acid Using Whole Cells of Recombinant Escherichiacoli with Separately and Coexpressed D−Hydantoinase and N-Carbamoylase

We developed a fully enzymatic process employing D−hydantoinase and N‐carbamoylase for the production of D−amino acid from 5′ ‐monosubstituted hydantoin. For the comparison of the reaction systems using two sequential enzymes, D−hydantoinase of Bacillus stearothermophilus SD1 and N‐carbamoyl‐D−amino acid amidohydrolase (N‐carbamoylase) of Agrobacterium tumefaciens NRRL B11291 were separately expressed in each host cell and coexpressed in the same host cell. A high level and constitutive expression of both enzymes in Escherichia coli in a soluble form was achieved using a promoter derived from B. stearothermophilus SD1. The expression levels of both enzymes ranged from 17% to 23% of the total soluble protein, depending on the expression system. In the case of employing separately expressed enzymes, the product yield of D−hydroxyphenylglycine from D,L−p‐hydroxyphenylhydantoin and productivity were 71% and 2.57 mM/g‐cell/h in 15 h, respectively. The accumulation of N‐carbamoyl‐D−hydroxyphenylglycine was significant over the reaction time. On the other hand, use of coexpressed enzymes resulted in 98% product yield of D−hydroxyphenylglycine in 15 h, minimizing the level of intermediates in the reaction mixture. The productivity of coexpressed whole cell reaction was estimated to be 6.47 mM/g‐cell/h in 15 h. The coexpressed system was tested for an elevated concentration of D,L−p‐hydroxyphenylhydantoin, and efficient production can be achieved.