Expression, purification, and characterization of a recombinant neutral ceramidase from mycobacterium tuberculosis

Nozomu Okino, Rie Ikeda, Makoto Ito

研究成果: Contribution to journalArticle査読

13 被引用数 (Scopus)

抄録

Ceramidase (CDase) catalyzes the hydrolysis of ceramide (Cer) to sphingosine (Sph) and fatty acid. We have reported the molecular cloning and preliminary characterization of the Mycobacterium CDase (MtCDase) (J. Biol. Chem., 274, 36616-36622 (1999)). To determine its function further, MtCDase was expressed in Escherichia coli and purified by Ni-Sepharose and gelfiltration. The purified recombinant enzyme showed a single band and a molecular weight estimated to be 71 kDa on SDS-PAGE. It had a pH optimum at 8.0-9.0 and quite broad specificity for various Cers. Of the Cers of different fatty acid moieties tested, those composed of C6-C24 fatty acids were well hydrolyzed, and Cers with mono unsaturated fatty acids were much more hydrolyzed than those with saturated fatty acids. Using N-dodecanoyl-7-nitrobenz-2-oxa-1,3-4- diazole (NBD)-d-erythro-sphingosine (C12-NBD-Cer) as substrates, the reaction followed normal Michaelis-Menten kinetics. The apparent Km and Vmax values for C12-NBD-Cer were 98.7 μM and 21.1 pmol/min respectively. The purified enzyme also catalyzed the synthesis of Cer in vitro, using NBD-labeled dodecanoic acid and Sph as substrates.

本文言語英語
ページ(範囲)316-321
ページ数6
ジャーナルBioscience, Biotechnology and Biochemistry
74
2
DOI
出版ステータス出版済み - 2010

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Analytical Chemistry
  • Biochemistry
  • Applied Microbiology and Biotechnology
  • Molecular Biology
  • Organic Chemistry

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