After hydrolyzed byβ-glucosidase, PNPG turn to a yellow product called p-Nitrophenol. Thus, β-glucosidase activity can be measured by determining the hydrolysis of p-nitrophenyl-β-D-glucopyranoside using the initial rate of colored reaction product accumulation.

The solid could be stored at room temperature.

Protocol 1. Prepare pNPG working solution at 5 mM in 50 mM sodium citrate buffer (pH 7.0). 2. Mix 20 μl aliquot of the acquired enzyme with 180 μL of pNPG working solution. 3. Incubate the mixture at 50 °C for 10 min. 4. Stop the reaction by adding 100 μl of ice-cold 0.5 M Na₂CO₃. 5. Detect the result at 405 nm with a Microplate Spectrophotometer. One unit (U) of β-glucosidase activity was defined as the amount of enzyme that released 1 μmol of pNP per minute from the substrate.{{Zhang L, Fu Q, Li W, Wang B, Yin X, Liu S, Xu Z, Niu Q. Identification and characterization of a novel β-glucosidase via metagenomic analysis of Bursaphelenchus xylophilus and its microbial flora. Sci Rep. 2017 Nov 1;7(1):14850. doi: 10.1038/s41598-017-14073-w. PMID: 29093477; PMCID: PMC5665999.|https://pubmed.ncbi.nlm.nih.gov/29093477/}}

After hydrolyzed byβ-glucosidase, PNPG turn to a yellow product called p-Nitrophenol. Thus, β-glucosidase activity can be measured by determining the hydrolysis of p-nitrophenyl-β-D-glucopyranoside using the initial rate of colored reaction product accumulation.