TY - JOUR
T1 - Expression, purification, and characterization of a recombinant neutral ceramidase from mycobacterium tuberculosis
AU - Okino, Nozomu
AU - Ikeda, Rie
AU - Ito, Makoto
N1 - Funding Information:
This work was supported in part by a Grant-in-Aid for Young Scientists B 19770086 (to N.O.) from The Ministry of Education, Culture, Sports, Science, and Technology, Japan.
PY - 2010
Y1 - 2010
N2 - 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.
AB - 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.
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U2 - 10.1271/bbb.90645
DO - 10.1271/bbb.90645
M3 - Article
C2 - 20139604
AN - SCOPUS:77249137735
VL - 74
SP - 316
EP - 321
JO - Bioscience, Biotechnology and Biochemistry
JF - Bioscience, Biotechnology and Biochemistry
SN - 0916-8451
IS - 2
ER -
