Abstract

A bioluminescent assay method for detecting the activity of phospholipase C (PLC; phosphatidyl choline cholinephosphohydrolase, EC 3.1.4.3) was developed using bioluminescent marine bacteria. Phospholipase C from Bacillus cereus and sn-1,2-dimyristoyl phosphatidyl choline (DMPC) as a substrate were used in the demonstration, and the produced sn-1,2-dimyristoyl glycerol was further hydrolyzed with lipase from Candida cylidracea. The hydrolyzed myristic acid was quantified using a dark mutant of Vibrio harveyi (designated as M-17). The in vivo light intensity of which was stimulated specifically up to one thousand fold in the presence of myristic acid. The rates of the hydrolysis of the DMPC substrate by the phospholipase measured by the luminescence method were linear with time and the amount of enzyme added. Activity measurement conditions (at 25 C, pH 6.5, 10 min fixed time assay) were established to detect as little as 0.1 mUnit of phospholipase C and 5 nM of myristic acid production.

• Keywords: Phospholipase C (PLC); bioluminescence; sn-1;2-dimyristoyl phosphatidyl choline (DMPC); dia-cyl glyc