Leucyl-aminopeptidase (LAP) and -1,3-glucanase activity assays
For protein extraction, fifty milligrams of fresh plant material were
extracted in the extraction buffer (50 mM TRIS-HCl, 0.5 mM
MnCl2, pH 8). One mL of the extraction buffer was added
to each sample and were centrifuge for 20 minutes at 10,000 g ,
4ºC. The supernatant was recovered and stored at -20ºC. For LAP
activity, Leu-p-nitroanilide (Sigma) was prepared from the stock
solution as enzyme substrate at a concentration of 3 mM in a solution of
50 mM TRIS-MnHCl2. The stock solution was previously
prepared at 150 mM in ethanol and stored at -20ºC. To carry out the
analysis, 40 µL of the protein sample and 200 µL of the substrate were
incubated for 15 minutes at 37ºC and absorbance was measured at 410 nm
as described in (Chao et al., 2000). β-1,3 glucanase activity was
measured by the Somogy-Nelson method as described by Roman and
co-workers (de Roman et al., 2011).