Title
Molecular cloning and characterization of the alkaline ceramidase from Pseudomonas aeruginosa PA01
Author
Nieuwenhuizen, W.F.
van Leeuwen, S.
Jack, R.W.
Egmond, M.R.
Götz, F.
Publication year
2003
Abstract
Ceramidase (CDase) hydrolyzes the amide bond in ceramides to yield free fatty acid and sphingosine. From a 3-L Pseudomonas aeruginosa PA01 culture, 70 μg of extracellular alkaline, Ca2+-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 DH5α and E. coli BL21 cells under control of the lac-promoter and T7-promoter, respectively. From a 3-L E. coli DH5α culture, 280 μg 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, 1.8mg 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. © 2003 Elsevier Science (USA). All rights reserved.
Subject
Biology
Food technology
Ceramidase
Gene
Inclusion body
Pseudomonas
Virulence factor
Amidohydrolases
Amino Acid Sequence
Base Sequence
Cloning, Molecular
Electrophoresis, Agar Gel
Inclusion Bodies
Models, Molecular
Molecular Sequence Data
Pichia
Protein Renaturation
Protein Sorting Signals
Pseudomonas aeruginosa
Recombinant Proteins
Sequence Analysis, Protein
Escherichia coli
Eukaryota
Pichia
Pichia pastoris
Prokaryota
Pseudomonas
Pseudomonas aeruginosa
Pseudomonas putida
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DOI
https://doi.org/10.1016/s1046-5928(03)00099-8
TNO identifier
237176
ISSN
1046-5928
Source
Protein Expression and Purification, 30 (1), 94-104
Document type
article