Screening, overexpression and characterization of an N -acylamino acid racemase from Amycolatopsis orientalis subsp. lurida

Abstract

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.

Enzymatic Racemisation and its Application to Synthetic Biotransformations

“…lurida [91] were found to be similar to those from Amycolatopsis sp. TS-1-60 and S. atratus, [95] i.e., a pI of 4.4, pH optimum of about 8, and thermal stability at 50 8C for 30 min.…”

“…lurida have gained importance. [91] By comparing the activity of NAAR from A. orientalis and Amycolatopsis sp. TS-1-60, a significant difference Scheme 12.…”

Structural Basis for Catalytic Racemization and Substrate Specificity of an N-Acylamino Acid Racemase Homologue from Deinococcus radiodurans

“…[5][6][7] Thus, these results together suggest a possible link between sequence and quaternary structure of the superfamily. One possible function of the higher level quaternary structure is that the intersubunit residues might participate in the catalytic reaction; however the structures show that the catalytic site is not in the intersubunit interface.…”

“…lurida and Amycolatopsis azurea were cloned and expressed in Escherichia coli. [4][5][6][7] Biochemical characterization shows that NAAAR acts on a broad range of N-acylamino acids rather than amino acids and that it requires a divalent metal ion for its activity, distinct from pyridoxal 5 0 -phosphate-dependent amino acid racemases. E-mail addresses of the corresponding authors: wcwang@life.nthu.edu.tw; whhsu@dragon.nchu.edu.tw Immobilization of NAAAR-aminoacylase in a bioreactor indeed allowed the continuous production of optically active methionine.…”

Rational re-design of the “double-racemase hydantoinase process” for optically pure production of natural and non-natural l-amino acids

“…An alternative method to produce optically pure amino acids is the "acylase process", where starting from racemic mixtures of Nacetyl-amino acids, an N-acetyl-amino acid racemase [29] (NAAAR; re-assigned as N-succinyl-amino acid racemase, NSAAR [30] is coupled with a d-or l-aminoacylase. In a previous work, we confirmed that a recombinant NSAAR from G. kaustophilus CECT4264 (GkNSAAR)), with substrate promiscuity, also racemized d-and l-N-carbamoyl-amino acids [31] .…”