Efficient conversion of 5-substituted hydantoins to D-α-amino acids using recombinant Escherichia coli strains

Abstract

D-Amino acids, important intermediates in the production of semisynthetic penicillins and cephalosporins, are currently prepared from the corresponding hydantoins using bacterial biomass containing two enzymes, hydantoinase and carbamylase. These enzymes convert the hydantoins first into carbamyl derivatives and then into the corresponding D-amino acids. In an attempt to select more efficient biocatalysts, the hydantoinase and carbamylase genes from Agrobacterium tumefaciens (formerly A. radiobacter) were cloned in Escherichia coli. The genes were assembled to give two operon-type structures, one having the carbamylase gene preceding the hydantoinase gene and the other with the carbamylase gene following the hydantoinase gene. The recombinant strains stably and constitutively produced the two enzymes and efficiently converted the corresponding hydantoins into p-hydroxyphenylglycine and phenylglycine. The order of the genes within the operon and the growth temperature of the strains turned out to be important for both enzyme and D-amino acid production. The configuration with the carbamylase gene preceding the hydantoinase gene was the most efficient one when the biomass was grown at 25 "C rather than 37 "C. This biomass produced D-amino acid twice as efficiently as the industrial strain of A. tumefaciens. The efficiency was found to be correlated with the level of carbamylase produced, indicating that the concentration of this enzyme is the rate-limiting factor in o-amino acid production under the conditions used on an industrial scale.

Construction and Evaluation of a Novel Bifunctional N -Carbamylase– d -Hydantoinase Fusion Enzyme

“…These amino acids currently are produced via two sequential reactions mediated by D-hydantoinase and N-carbamylase (29,30). For process development, each enzyme has been separately expressed in different host cells (4,12,17,18) or in a single host (8). In this process, the different expression levels of the two each enzymes, the formation of inclusion bodies, and the transport of restricted substrates through the cell membrane remained to be solved.…”

“…Both CAB-HYD and CAB-HYD1 fusion enzymes also required a reducing agent such as DTT for the N-carbamylase activity (7,8). The fusion enzymes lost the N-carbamylase activity more readily than the D-hydantoinase activity.…”

“…Due to the great commercial demand for various D-amino acids, enzymatic and chemoenzymatic routes have been developed (29). Of these, a fully enzymatic process using two sequential enzymes, D-hydantoinase and N-carbamylase, is a typical case requiring combined enzyme activity (8). In this process, hydantoin derivative is hydrolyzed by D-hydantoinase, and the resulting N-carbamyl-D-amino acid is further converted to the corresponding D-amino acid by N-carbamylase.…”

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

“…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 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.…”