In all cases, enzyme samples of 10 μL, hydro distillate extract from the cell suspension after 40 days, and pure compounds (thymol, estragole, p-cymene, γ-terpinene, linalool, β-terpineol, ocimene, eugenol, 1,8-cineole, β-Caryophyllene, and germacrene D) (50, 100, 500, 1000, and 5000 mg/L) were pre-incubated together for 10 min. Substrate was then added to start the reaction (20 min for Leupeptin). The reaction lasted for 180 min at 37 °C and was then stopped using 300 µL of cold 10% (v/v) trichloroacetic acid (TCA). The reaction mixture was centrifuged at 5000× g for 20 min using the Sigma 3k30 cooling centrifuge. Ten microliters of NaOH (10 N) was added to the supernatant, and absorbency at 450 nm was measured using an ELISA plate reader. An assay mixture without an enzyme was used as a blank, the specific activity of total proteolytic enzymes was calculated as OD450. mg−1·protein−1·h−1, and a blank sample was determined without an enzyme solution.
Proteolytic Enzyme Activity Assay in Red Palm Weevil Larvae
In all cases, enzyme samples of 10 μL, hydro distillate extract from the cell suspension after 40 days, and pure compounds (thymol, estragole, p-cymene, γ-terpinene, linalool, β-terpineol, ocimene, eugenol, 1,8-cineole, β-Caryophyllene, and germacrene D) (50, 100, 500, 1000, and 5000 mg/L) were pre-incubated together for 10 min. Substrate was then added to start the reaction (20 min for Leupeptin). The reaction lasted for 180 min at 37 °C and was then stopped using 300 µL of cold 10% (v/v) trichloroacetic acid (TCA). The reaction mixture was centrifuged at 5000× g for 20 min using the Sigma 3k30 cooling centrifuge. Ten microliters of NaOH (10 N) was added to the supernatant, and absorbency at 450 nm was measured using an ELISA plate reader. An assay mixture without an enzyme was used as a blank, the specific activity of total proteolytic enzymes was calculated as OD450. mg−1·protein−1·h−1, and a blank sample was determined without an enzyme solution.
Corresponding Organization : King Faisal University
Other organizations : Minia University
Protocol cited in 2 other protocols
Variable analysis
- Concentration of pure compounds (thymol, estragole, p-cymene, γ-terpinene, linalool, β-terpineol, ocimene, eugenol, 1,8-cineole, β-Caryophyllene, and germacrene D) (50, 100, 500, 1000, and 5000 mg/L)
- Total proteolytic enzyme activity
- Protein concentration
- Assay buffer composition (50 mM HEPS (N-2-hydroxyethyl piperazin-N'-2-ethanesulphonic acid), pH 8.0, 5 mM dithiothreitol (DTT) and 0.1% (v/v) Triton X-100)
- Incubation time (20 min for Leupeptin, 180 min for other compounds)
- Incubation temperature (37 °C)
- Positive control: Enzyme samples of 10 μL
- Negative control: Assay mixture without an enzyme
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