Optically pure D- or L-amino acids are used as intermediates in several industries. D-amino acids are involved in the synthesis of antibiotics, pesticides, sweeteners and other biologically active peptides. L-amino acids are used as feed and food additives, as intermediates for pharmaceuticals, cosmetics, pesticides and as chiral synthons in organic synthesis. The specific activity of these optically pure amino acids depends on their structure, chirality and purity. There are two main approaches to obtain optically pure amino acids, namely chemical and enzymatic synthesis. Chemical synthesis gives racemic mixtures of amino acids of low yield and is not environment friendly. One of the most widely-used enzymatic method is the “Hydantoinase Process”. In this cascade of reactions, the chemically synthesized D,L-5-monosubstituted hydantoin ring is first hydrolyzed by a stereoselective hydantoinase enzyme to give the corresponding N-carbamoyl α-amino acid that is hydrolyzed by highly enantiospecific N-carbamoyl α-amino acid amidohydrolase (Ncarbamoylase) to yield the free amino acid. At the same time, the remaining non-hydrolyzed 5-monosubstituted hydantoin is racemized by the hydantion racemase enzyme. This process has evolved over the years from the isolation of microorganisms with one or several of these enzymes to the construction of recombinant systems for industrial application.

A reaction system was developed for the production of d ‐amino acids from d , l ‐5‐monosubstituted hydantoins with a very slow rate of spontaneous racemization. For this purpose the d ‐hydantoinase and d ‐carbamoylase from Agrobacterium radiobacter NRRL B11291 were cloned in separate plasmids and expressed in Escherichia coli . The third enzyme, hydantoin racemase, was cloned from Agrobacterium tumefaciens C58. The hydantoin racemase amino acid sequence was significantly similar to those previously described. A reaction system consisting of recombinant Escherichia coli whole cell biocatalysts containing separately expressed d ‐hydantoinase, d ‐carbamoylase, and hydantoin recemase showed high substrate specificity and was effective toward both aliphatic and aromatic d , l ‐5‐monosubstituted hydantoins. After optimizing reaction conditions (pH 8 and 50 °C), 100% conversion of d , l ‐5‐(2‐methylthioethyl)‐hydantoin (15 mM) into d ‐methionine was obtained in 30 min.