Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 mug of extracellular alkaline, Ca2+-dependent Mase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The Mase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. Mase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular Mase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated Mase were expressed. High levels of intracellular CDase were expressed by Escherichia coli DH5alpha and E coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E coli DH5alpha culture, 280 mug of pure Mase was obtained after a three-step purification protocol. Under control of the T7-promotor Mase, without its signal peptide, was produced in inclusion bodies in E coli BL21 cells. After refolding, 1.8 mg of pure active Mase was obtained from a 2.4-L culture after ammonium sulfate precipitation and gel filtration. Both the recombinant and wild-type, CDases have a pH optimum of 8.5. The recombinant enzyme was partially characterized. This is the first report of a high yield Mase production system allowing detailed characterization of the enzyme at the molecular level. (C) 2003 Elsevier Science (USA). All rights reserved.