Screening, overexpression and characterization of an N -acylamino acid racemase from Amycolatopsis orientalis subsp. lurida
| Autor: | M.-R. Kula, S Verseck, Andreas S. Bommarius |
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| Rok vydání: | 2001 |
| Předmět: |
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Genetic Vectors Molecular Sequence Data EcoRI Isomerase medicine.disease_cause Applied Microbiology and Biotechnology Substrate Specificity Actinomycetales Escherichia coli medicine Edetic Acid Amycolatopsis orientalis Amino Acid Isomerases chemistry.chemical_classification Molecular mass biology General Medicine biology.organism_classification Molecular biology Recombinant Proteins Amino acid Molecular Weight Isoelectric point Enzyme Biochemistry chemistry Metals biology.protein Hydroxymercuribenzoates Electrophoresis Polyacrylamide Gel Biotechnology |
| Zdroj: | Applied Microbiology and Biotechnology. 55:354-361 |
| ISSN: | 1432-0614 0175-7598 |
| DOI: | 10.1007/s002530000508 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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