PNPG (4-Nitrophenyl β-D-glucopyranoside) is a chromogenic substrate for β-D-glucosidase that, upon enzymatic cleavage, yields a yellow solution.

I. β-D-glucosidase activity assay
1. Material preparation:
1) PNPG solution: Dissolve PNPG in an appropriate amount of buffer, usually at a concentration of 1-5 mM. The solution is usually prepared using phosphate buffer (PBS, pH 7.0-7.5).
2) Enzyme solution: Purified β-D-glucosidase or cell extract, enzyme concentration adjusted according to experimental requirements.
3) Buffer: Usually use phosphate buffer (PBS, pH 7.0-7.5).
2. Experimental steps:
1) Prepare PNPG solution and add it to the reaction tube.
2) Add an appropriate amount of β-D-glucosidase solution (usually within the concentration range of the enzyme) and mix well.
3) Incubate the reaction system at 37°C for a certain time (usually 10-30 minutes).
4) After the reaction is completed, add a stop solution (such as NaOH solution) to stop the reaction. The yellow color change of the solution indicates that the reaction is complete.
5) Use a spectrophotometer to measure the absorbance value at 405 nm. The absorbance is proportional to the activity of the enzyme.
2. Application in enzyme inhibitor screening
When adding different concentrations of potential inhibitors, measuring the change in enzyme activity can be used to screen inhibitors of β-D-glucosidase.