Purification, characterization and regulation of a monomeric L-phenylalanine dehydrogenase from the facultative methylotroph Nocardia sp. 239

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Abstract

In Nocardia sp. 239 D-phenylalanine is converted into L-phenylalanine by an inducible amino acid racemase. The further catabolism of this amino acid involves an NAD-dependent L-phenylalanine dehydrogenase. This enzyme was detected only in cells grown on L- or D-phenylalanine and in batch cultures highest activities were obtained at relatively low amino acid concentrations in the medium. The presence of additional carbon- or nitrogen sources invariably resulted in decreased enzyme levels. From experiments with phenylalanine-limited continuous cultures it appeared that the rate of synthesis of the enzyme increased with increasing growth rates. The regulation of phenylalanine dehydrogenase synthesis was studied in more detail during growth of the organism on mixtures of methanol and L-phenylalanine. Highest rates of L-phenylalanine dehydrogenase production were observed with increasing ratios of L-phenylalanine/methanol in the feed of chemostat cultures. Characteristic properties of the enzyme were investigated following its (partial) purification from L- and D-phenylalanine-grown cells. This resulted in the isolation of enzymes with identical properties. The native enzyme had a molecular weight of 42000 and consisted of a single subunit; it showed activity with L-phenylalanine, phenylpyruvate, 4-hydroxyphenylpyruvate, indole-3-pyruvate and α-ketoisocaproate, but not with imidazolepyruvate, D-phenylalanine and other L-amino acids tested. Maximum activities with phenylpyruvate (310 µmol min-1 mg-1 of purified protein) were observed at pH 10 and 53°C. Sorbitol and glycerol stabilized the enzyme.
Original languageEnglish
Pages (from-to)12-18
Number of pages7
JournalArchives of Microbiology
Volume153
Issue number1
DOIs
Publication statusPublished - Dec-1989

Keywords

  • Continuous culture
  • Regulation
  • Methanol
  • Phenylalanine
  • Phenylalanine dehydrogenase
  • Nocardia sp. 239

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