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

• Published in: Applied microbiology and biotechnology Vol. 55; no. 3; pp. 354 - 361
• Main Authors: Verseck, S, Bommarius, A, Kula, M.R
• Format: Journal Article
• Language: English
• Published: Germany Springer Nature B.V 01.04.2001
• Subjects: aar gene; Actinomycetales - enzymology; Actinomycetales - genetics; Amino Acid Isomerases - chemistry; Amino Acid Isomerases - genetics; Amino Acid Isomerases - metabolism; Amino acids; Amycolatopsis orientalis lurida; Amycolatopsis orientalis subsp. lurida; anion exchange chromatography; Bacteria; Cloning; cobalt; Deoxyribonucleic acid; DNA; DNA fragmentation; DNA sequencing; E coli; Edetic Acid - pharmacology; EDTA (chelating agent); Electrophoresis, Polyacrylamide Gel; Enzymes; Escherichia coli; Escherichia coli - genetics; gel chromatography; genes; Genetic screening; Genetic Vectors; Heat treatment; Hot Temperature; Hydroxymercuribenzoates - pharmacology; isoelectric point; magnesium; manganese; metal ions; Metals - pharmacology; Molecular Sequence Data; Molecular Weight; N-Acylamino acid racemase; polyacrylamide gel electrophoresis; polymerase chain reaction; protein subunits; Proteins; racemase; Recombinant Proteins - biosynthesis; screening; Southern blotting; Substrate Specificity
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s002530000508

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.