X-Neu5Ac: a novel substrate for chromogenic assay of neuraminidase activity in bacterial expression systems.
A chromogenic substrate 1, 5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-alpha-D-glycero-D-galacto-2-nonulopyranosidon ic acid (X-Neu5Ac), has been synthesized to facilitate the screening of bacterial colonies or plaques for the detection of either natural or mutant neuraminidase activity. Substrate 1 was hydrolyzed by neuraminidase isolated from Clostridium perfringens to release a halogenated indol-3-ol 2 that undergoes rapid aerobic oxidation to form the dark blue pigment, 5,5'-dibromo-4,-4'-dichloroindigo 3. Preliminary kinetic studies indicate that this compound is a good substrate (Km 0.89 x 10(-3) M) for neuraminidase and is quite stable under identical conditions in the absence of enzyme. These results suggest that X-Neu5Ac 1 can be useful to screen for bacterially-encoded enzyme production directly on agar plates.
Fujii
I
I
Protein Engineering Research Institute, Osaka, Japan.
Iwabuchi
Y
Y
Teshima
T
T
Shiba
T
T
Kikuchi
M
M
D002863:Chromogenic Compounds / Q000138:chemical synthesis* / Q000378:metabolism; D003016:Clostridium perfringens / Q000201:enzymology* / Q000235:genetics; D006868:Hydrolysis; D007211:Indoles / Q000138:chemical synthesis* / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation / Q000235:genetics; D009438:Neuraminic Acids / Q000138:chemical synthesis* / Q000378:metabolism; D009439:Neuraminidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013379:Substrate Specificity
C089351:5-bromo-4-chloroindol-3-yl 5-acetamido-3,5-dideoxy-glycero-galacto-2-nonulopyranosidonic acid; D002863:Chromogenic Compounds; D007211:Indoles; D009438:Neuraminic Acids; D009439:Neuraminidase