反应详情

A p-nitrophenylferulate stock solution was made by dissolving p-nitrophenylferulate in dimethylsulfoxide (DMSO) to constitute a 0.1 M solution. Before assay, a sample of the stock solution was diluted 100-fold in 50 mM sodium acetate pH 5.0 to make a 1 mM solution. A 100 μl volume of 1 mM p-nitrophenylferulate was mixed with each dilution of the enzyme to give a 200 □l total volume, and then incubated at 25° C. for 10 minutes. Substrate alone, enzyme alone, and buffer alone were run as controls. p-Nitrophenol standard solutions of 0.25, 0.2, 0.1, 0.05, and 0.02 mM were prepared by diluting a 10 mM stock solution in 50 mM sodium acetate pH 5.0. At 10 minutes, 50 μl of 1.0 M Tris-HCl pH 8.0 buffer was added to each well (including samples, substrate control, enzyme control, reagent control, and standards), mixed, and the absorbance at 405 nm and 540 nm immediately measured on a SPECTRAMAX™ 340 PC plate reader (Molecular Devices, Sunnyvale, Calif., USA). The absorbance at 405 nm was corrected for the background absorbance at 540 nm from the insoluble substrate material. The activity measured was 14 Units per mg of protein. One unit of feruloyl esterase activity was defined as the amount of enzyme capable of releasing 1 μmole of p-nitrophenolate anion per minute at pH 5, 25° C.

1. customto give a 200 □l total volume
2. waitAt 10 minutes
3. additionmixed
4. customat pH 5, 25° C.