Production of D-amino acid using whole cells of recombinant Escherichia coli with separately and coexpressed D-hydantoinase and N-carbamoylase

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We developed a fully enzymatic process employing D-hydantoinase and N-carbamoylase for the production of D-amino aid 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.
Publisher
AMER CHEMICAL SOC
Issue Date
2000
Language
English
Article Type
Article
Keywords

THERMOSTABLE D-HYDANTOINASE; HETEROGENEOUS REACTION SYSTEM; D-P-HYDROXYPHENYLGLYCINE; IN-VITRO; AMIDOHYDROLASE; PROTEIN; CONVERSION; EXPRESSION; EVOLUTION; STRAINS

Citation

BIOTECHNOLOGY PROGRESS, v.16, no.4, pp.564 - 570

ISSN
8756-7938
DOI
10.1021/bp0000611
URI
http://hdl.handle.net/10203/14302
Appears in Collection
BS-Journal Papers(저널논문)
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