ONPG is a synthetic analog of lactose
used in biochemical assays to detect β-galactosidase activity. The enzyme hydrolyses the colourless ONPG into galactose
and orto-nitrophenol (figure 1), which is yellow in colour with maximum absorbance at 420 nm in alkaline conditions.
Figure 1. ONPG hydrolysis by β-galactosidase.
Detection and quantification of β-galactosidase activity
ONPG is used as chromogenic substrate for quantitative studies and enzyme kinetics of β-galactosidase. The reaction can be stopped with sodium carbonate
and absorbance read at 420 nm.
ONPG test in diagnostics
This test is routinely used in diagnostics for the identification of lactose-fermenting bacteria of the Enterobacteriaceae family. The ability to ferment lactose is predetermined by the presence of two key proteins; a permease, which is a membrane protein that transports lactose (and its analogues) into the cell, and β-galactosidase, which is a cytoplasmic enzyme that hydrolyses lactose. E. coli
spp. and Enterobacter
spp. possess both proteins and are ONPG positive. Members of Citrobacter
spp. and Arizona
spp. lack permease and are ONPG negative, as well as β-galactosidase negative bacteria Salmonella