Purification and characterization of a novel ceramidase from pseudomonas aeruginosa

Nozomu Okino, Motohiro Tani, Shuhei Imayama, Makoto Ito

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We report here a novel type of ceramidase of Pseudomonas aeruginosa AN17 isolated from the skin of a patient with atopic dermatitis. The enzyme was purified 83,400-fold with an overall yield of 21.1% from a culture supernatant of strain AN17. After being stained with a silver staining solution, the purified enzyme showed a single protein band, and its molecular mass was estimated to be 70 kDa on SDS-polyacrylamide gel electrophoresis. The enzyme showed quite wide specificity for various ceramides, i.e. it hydrolyzed ceramides containing C12:0-C18:0 fatty acids and 7-nitrobenz-2- oxa-1,3-4-diazole-labeled dodecanoic acid, and not only ceramide containing sphingosine (d18:l) or sphinganine (d18:0) but also phytosphingosine (t18:0) as the long-chain base. However, the enzyme did not hydrolyze galactosylceramide, sulfatide, GM1, or sphingomyelin, and thus was clearly distinguished from a Pseudomonas sphingolipid ceramide N-deacylase (Ito, M., Kurita, T., and Kita, K. (1995) J. Biol. Chem. 270, 24370-24374). This bacterial ceramidase had a pH optimum of 8.0-9.0, an apparent K(m) of 139 μM, and a V(max) of 5.3 μmol/min/mg using N-palmitoylsphingosine as the substrate. The enzyme appears to require Ca2+ for expression of the activity. Interestingly, the 70-kDa protein catalyzed a reversible reaction in which the N-acyl linkage of ceramide was either cleaved or synthesized. Our study demonstrated that ceramidase is widely distributed from bacteria to mammals.

Original languageEnglish
Pages (from-to)14368-14373
Number of pages6
JournalJournal of Biological Chemistry
Issue number23
Publication statusPublished - Jun 5 1998

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology


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