title: Bioluminescent assay for sphingolipid ceramide N-deacylase using Vibrio harveyi dark mutant M-17.
abstract: A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC 3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:l) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 microU and 5 nM of myristic acid production were detected in less than one min. The assay worked well for the determination of Km and chromatographic fraction assay.
authors.affiliation: Department of Marine Biotechnology, Anyang University, Incheon 417-833, Republic of Korea. kwcho@anyang.ac.kr
authors.forename: Ki Woong
authors.initials: KW
authors.lastname: Cho
mesh_terms: D000581:Amidohydrolases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism*; D002518:Ceramides / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods*; D009154:Mutation; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D002518:Ceramides; D000581:Amidohydrolases; C096410:sphingolipid ceramide N-deacylase