Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01.
Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 microg of extracellular alkaline, Ca(2+)-dependent CDase, was purified to homogeneity, the N-terminal sequence was determined, and the CDase gene was cloned. The CDase gene encodes a 670 amino acid protein with a 26 amino acid signal peptide. CDase was expressed in five prokaryotic and eukaryotic expression systems. Small amounts of recombinant active extracellular CDase were expressed by Pseudomonas putida KT2440. In Pichia pastoris GS115 low amounts of recombinant extracellular glycosylated CDase 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 microg of pure CDase was obtained after a three-step purification protocol. Under control of the T7-promotor CDase, without its signal peptide, was produced in inclusion bodies in E. coli BL21 cells. After refolding, 1.8 mg of pure active CDase 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 CDase production system allowing detailed characterization of the enzyme at the molecular level.
Nieuwenhuizen
Willem F
WF
Microbial Genetics, University of Tübingen, Waldhäuser Strasse 70/8, 72076 Tübingen, Germany. nieuwenhuizen@voeding.tno.nl
van Leeuwen
Sander
S
Jack
Ralph W
RW
Egmond
Maarten R
MR
Götz
Friedrich
F
D000581:Amidohydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D055572:Ceramidases; D003001:Cloning, Molecular; D004587:Electrophoresis, Agar Gel; D002479:Inclusion Bodies / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics; D020673:Protein Renaturation; D021382:Protein Sorting Signals / Q000235:genetics / Q000502:physiology; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics* / Q000378:metabolism*; D020539:Sequence Analysis, Protein
D021382:Protein Sorting Signals; D011994:Recombinant Proteins; D000581:Amidohydrolases; D055572:Ceramidases