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.