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Purification and properties of D-aspartate oxidase from Cryptococcus humicolus UJ1.

Authors
  • Yamada, R
  • Ujiie, H
  • Kera, Y
  • Nakase, T
  • Kitagawa, K
  • Imasaka, T
  • Arimoto, K
  • Takahashi, M
  • Matsumura, Y
Type
Published Article
Journal
Biochimica et Biophysica Acta
Publisher
Elsevier
Publication Date
May 23, 1996
Volume
1294
Issue
2
Pages
153–158
Identifiers
PMID: 8645733
Source
Medline
License
Unknown

Abstract

D-Aspartate oxidase (EC 1.4.3.1), which is highly specific to D-aspartate, was inducibly produced by a yeast strain which was isolated from soil and identified as Cryptococcus humicolus UJ1. The enzyme was purified to homogeneity as indicated on SDS-polyacrylamide gel electrophoresis. The molecular mass of the monomer subunit was determined to be 40 kDa. The native enzyme was suggested to be a homotetramer by its behavior on gel filtration. The enzyme was shown to be a flavoprotein by its absorption spectral properties, and the flavin was found to be tightly, but not covalently, bound FAD. The purified preparation had a specific activity of 76.1 mumol/min per mg protein with D-aspartate as substrate. Optimum pH was 7.5 and optimum temperature was around 35 degrees C. D-Glutamate was a very poor substrate for the enzyme. N-Methyl-D-aspartate was better than D-glutamate as substrate but markedly poorer than D-aspartate. Malonate was the most effective competitive inhibitor of the compounds tested. The N-terminal amino-acid sequence of the enzyme showed a significant homology with those of D-aspartate oxidases from beef kidney and Octopus vulgaris and those of D-amino-acid oxidases from various sources.

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