Two different dynamic kinetic resolution processes for the production of a number of natural and nonnatural l-amino acids at 100% chemical and optical yield have recently been established at Degussa. The first process is based on the dynamic kinetic resolution of 5-monosubstituted hydantoins using tailor-made whole-cell biocatalysts coexpressing a l-carbamoylase, a hydantoin racemase, and a hydantoinase. The hydantoin-converting pathway was optimized by adjusting expression levels of the respective enzymes as well as by inverting the enantioselectivity of the d-selective hydantoinase. This resulted overall in a 50-fold improved productivity and significant reduction of biocatalyst cost. The second process is based on the dynamic kinetic resolution of N-acetyl amino acids using an acylase in combination with a novel racemase from Amycolatopsis orientalis subsp. lurida. This racemase could overcome the problem of substrate inhibition and requirement of high concentrations of divalent metal ions which limits the use of other N-acylamino acid racemases described in the literature.

Thirty-one different actinomycete strains were used in a genetic screening using PCR and Southern hybridization methods to detect N-acetylamino acid racemases (AAR) in order to obtain enzymes with different properties. Cloning and sequencing of a 2.5 kb EcoRI DNA fragment from Amycolatopsis orientalis subsp. lurida revealed the coding gene of an N-acetylamino acid racemase, which had identities to the aar gene of Amycolatopsis sp. TS-1-60 [Tokuyama and Hatano (1995) Appl Microbiol Biotechnol 42:884-889] of 86% at the level of DNA, and 90% at the level of amino acids. The heterologous overexpression in Escherichia coli resulted in a specific activity of about 0.2 U/mg of this racemase. A two-step purification with heat treatment followed by anion-exchange chromatography led to almost homogeneous enzyme. The optimum pH of the enzyme was 8.0 and it was stable at 50 degrees C for 30 min. The relative molecular mass of the native enzyme and the subunit was calculated to be 300 kDa and 40 kDa by gel filtration and SDS-PAGE, respectively. The isoelectric point (pI) of the AAR was 4.4. It catalyzed the racemization of optically active N-acetylamino acids such as N-acetyl-L- or -D-methionine and N-acetyl-L-phenylalanine. Further characterization of the racemase demonstrated a requirement for divalent metal ions (Co2+, Mn2+, Mg2+) for activity and inhibition by EDTA and p-hydroxymercuribenzoic acid. AAR is sensitive to substrate inhibition at concentrations exceeding 200 mM.