KMID : 0880220080460050585
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Journal of Microbiology 2008 Volume.46 No. 5 p.585 ~ p.589
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Bioluminescent Assay for Sphingolipid Ceramide N-Deacylase Using Vibrio harveyi Dark Mutant M-17
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Cho Ki-Woong
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Abstract
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A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:1) 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 ¥ìU 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.
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KEYWORD
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bioluminescent assay, Vibrio harveyi M-17, SCDase, ceramide, myristic acid
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