Water → Phenyl nitrate

Participants

Reaction Steps

After the culture, each 1 ml of culture medium was added 15 μl of toluene and shaked at 37° C. for 30 minutes. After shaking, the culture medium was diluted 10 to 100 times in order to measure the produced amount of β-galactosidase. One ml of the solution was taken out and shaked at 30° C. for 5 minutes, followed by addition of 3.5 ml of 0.2M phosphate buffer (pH 7.25) and 0.5 ml of 0.01M O-nitrophenyl-β-D-galactopyranoside (ONPG) and 10 minutes' shaking. After sampling 1 ml of the solution and stopping the reaction by placing it in 1M Na2CO3, 8 ml of water was added thereto so as to make the whole amount 10 ml. The absorbance of this sample was measured at 420 nm by using a spectrophotometer. The amount of O-nitrophenol (ONP) produced was calculated from the standard curves of ONT. In this case, the β-galactosidase activity for decomposing 1 μmole of ONPG per minute was defined as one unit (U).

Provenance & Evidence