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

Nozomu Okino, Rie Ikeda, Makoto Ito

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)316-321
Number of pages6
JournalBioscience, Biotechnology and Biochemistry
Volume74
Issue number2
DOIs
Publication statusPublished - Mar 1 2010

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Neutral Ceramidase
Mycobacterium tuberculosis
Purification
Ceramidases
Fatty Acids
lauric acid
Sphingosine
Ceramides
Mycobacterium
Monounsaturated fatty acids
Cloning
Molecular Cloning
Substrates
Enzymes
Unsaturated Fatty Acids
Sepharose
Escherichia coli
Polyacrylamide Gel Electrophoresis
Hydrolysis
Molecular Weight

All Science Journal Classification (ASJC) codes

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

Cite this

Expression, purification, and characterization of a recombinant neutral ceramidase from mycobacterium tuberculosis. / Okino, Nozomu; Ikeda, Rie; Ito, Makoto.

In: Bioscience, Biotechnology and Biochemistry, Vol. 74, No. 2, 01.03.2010, p. 316-321.

Research output: Contribution to journalArticle

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