Determination of the enzyme activity by DNS
The DNS method was used to detect the xylanase activity of the XynA and XynA E182A/E280A proteins. In this study, BWX (Lot# X4252, ≥ 90% purity) was used as the substrate, and xylose was used as the standard. The reducing sugars released from BWX were measured by DNS reagent to estimate the xylanase activity. The hydrolyzed product oligosaccharides (reducing sugars) undergo redox reactions with 3,5-dinitrosalicylic acid (DNS) to produce 3-amino-5-nitrosalicylic acid (brown red). First, we took the appropriate concentration (25, 250, 500, 1000 nM) of the protein and 0.5% (w/v) of BWX and mixed them evenly in 100 mM sodium citrate at pH 6.5, and the total volume was 50 μL. Second, the reaction system was incubated at the optimal temperature of 65 °C for 10 minutes. Third, the DNS reagent was added to the reaction system, and the mixture was incubated at 95 °C for 5 minutes. Finally, the reaction system was cooled in an ice bath to room temperature and diluted with 100 mM sodium citrate buffer solution at pH 6.5, and the absorbance was determined at 540 nm with an enzyme label analyzer.
Results