Production of D-amino acid using whole cells of recombinant Escherichia coli 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.
Park
J H
JH
Department of Biological Sciences, Korea Advanced Institute of Science and Technology, 373-1 Kusung-dong Yusong-gu, Taejon 305-701 Korea.
Kim
G J
GJ
Kim
H S
HS
D000581:Amidohydrolases / Q000235:genetics*; D000596:Amino Acids / Q000096:biosynthesis*; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D011995:Recombination, Genetic
D000596:Amino Acids; D017931:DNA Primers; D000581:Amidohydrolases; C080477:N-carbamoyl-D-amino acid amidohydrolase; C020047:dihydropyrimidinase