EFFICIENT CONVERSION OF 5-SUBSTITUTED HYDANTOINS TO D-ALPHA-AMINO ACIDS USING RECOMBINANT ESCHERICHIA-COLI STRAINS

D-Amino acids, important intermediates in the production of semisynthe tic penicillins and cephalosporins, are currently prepared from the co rresponding hydantoins using bacterial biomass containing two enzymes, hydantoinase and carbamylase. These enzymes convert the hydantoins fi rst into carbamyl derivatives and then into the corresponding D-amino acids. In an attempt to select more efficient biocatalysts, the hydant oinase and carbamylase genes from Agrobacterium tumefaciens (formerly A. radiobacter) were cloned in Escherichia coli. The genes were assemb led to give two operon-type structures, one having the carbamylase gen e preceding the hydantoinase gene and the other with the carbamylase g ene following the hydantoinase gene. The recombinant strains stably an d constitutively produced the two enzymes and efficiently converted th e corresponding hydantoins into p-hydroxyphenylglycine and phenylglyci ne. The order of the genes within the operon and the growth temperatur e of the strains turned out to be important for both enzyme and D-amin o acid production. The configuration with the carbamylase gene precedi ng the hydantoinase gene was the most efficient one when the biomass w as grown at 25 degrees C rather than 37 degrees C. This biomass produc ed D-amino acid twice as efficiently as the industrial strain of A. tu mefaciens. The efficiency was found to be correlated with the level of carbamylase produced, indicating that the concentration of this enzym e is the rate-limiting factor in D-amino acid production under the con ditions used on an industrial scale.