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

Abstract

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.

Crystal Structure ofd-Hydantoinase fromBurkholderia pickettiiat a Resolution of 2.7 Angstroms: Insights into the Molecular Basis of Enzyme Thermostability

“…The homologous enzymes in microorganisms are hydantoinases (HYDs) which catalyze the hydrolysis of 5-substituted hydantoins to the enantiomerically pure Ncarbamyl amino acids. The latter can be converted chemically or enzymatically to the corresponding optically pure amino acids (50,54,64). In the biotechnology industry, HYD is widely used in the production of D-amino acids, including D-p-hydroxyphenylglycine, which are the precursors for semisynthesis of antibiotics, peptide hormones, pyrethroids, and pesticides (3,49,53,54).…”

Recombinant Polycistronic Structure of Hydantoinase Process Genes in Escherichia coli for the Production of Optically Pure d -Amino Acids

“…Previous works have suggested that the D-carbamoylase enzyme step limits the overall process and consequently causes high accumulation of the intermediate (N-carbamoyl-D-amino acid) in the reaction (14,19). For this reason, the D-carbamoylase gene was cloned closest to the promoter in order to obtain the highest synthesis of D-carbamoylase among the three enzymes.…”

“…This method is both cheaper and less contaminating than the chemoenzymatic process (10). In this cascade of reactions, named the hydantoin process (1) (4,8,19), only 50% of the remaining hydantoins are converted to the corresponding amino acid, while the other 50% correspond to L-hydantoin, which is not hydrolyzed by D-hydantoinase. For these hydantoins, faster racemization is possible via an enzymatic reaction incorporating a third enzyme named hydantoin racemase together with D-hydantoinase and D-carbamoylase enzymes.…”

“…High velocities of chemical racemization have been observed only for D,L-5-phenyl-and D,L-5-p-hydroxy-phenylhydantoin because of the resonance stabilization by the 5-substituent, while all other hydantoins take many hours to racemize (13). Consequently, although the hydantoinase process has been successfully used to produce 100% D-phenyl-and D-p-hydroxy-phenylglycine (4,8,19), only 50% of the remaining hydantoins are converted to the corresponding amino acid, while the other 50% correspond to L-hydantoin, which is not hydrolyzed by D-hydantoinase. For these hydantoins, faster racemization is possible via an enzymatic reaction incorporating a third enzyme named hydantoin racemase together with D-hydantoinase and D-carbamoylase enzymes.…”

“…This strategy involves adding several antibiotics to the medium, resulting in high selective pressure on the growth of the recombinant cells. Additionally, both of these recombinant systems have shown intermediate accumulation as a result of weak D-carbamoylase activity, which has been described as a limiting factor in the overall process (19).…”