# Effects of Melatonin Treatment of Postharvest Pear Fruit on Aromatic Volatile Biosynthesis
## 4 Materials and Methods
### Enzyme Activity  
LOX enzymes were extracted from 0.1 g of fruit tissue ground in liquid nitrogen using polyvinylpolypyrrolidone (PVPP), to which 1 mL of enzyme extract (0.1 mM phosphate buffer, pH = 7.5, 2 mM DTT, 1 mM disodium EDTA, and 0.1% (_v/v_) Triton X-100) was added, and then the mixture was centrifuged for 15 min at 4 \({}^{\circ}\)C and 15,000\(\times\)_g_. The reaction system comprised 0.1 mM phosphate buffer (pH = 7.5), reaction substrate solution (8.6 mM linoleic acid and 0.25% (_v/v_) Tween-20 soluble in 0.1 mM phosphate buffer), and crude enzyme solution. Change in absorbance per min at OD\({}_{280}\) was recorded 15 s after the addition of the enzyme solution, where each change in OD value was denoted as 1 U [32].  
HPL activity was determined following a modified version of the approach described by [PERSON] [33], where 3 g of fruit tissue was treated with 6 mL of pre-cooled (4 \({}^{\circ}\)C) extracting solution that contained 150 mmol/L HEPES-KOH buffer (pH = 8.0), 250 mmol L\({}^{-1}\) sorbitol, 10 mmol L\({}^{-1}\) EDTA, 10 mmol L\({}^{-1}\) MgCl\({}_{2}\), 1% (_v/v_) glycerol, 4% PVPP, and 0.1 mmol L\({}^{-1}\) MPMSF (protease inhibitor). Then, samples were ground using a pre-chilled mortar and pestle, fully blended, and centrifuged at 15,000\(\times\)\(g\) (4 \({}^{\circ}\)C) for 30 min. HPL activity was determined using a 3.5 mL reaction system, comprising 2 mL of analytical buffer (150 mmol L\({}^{-1}\) HEPES-KOH, pH = 8.0; 250 mmol L\({}^{-1}\) sorbitol; 10 mmol L\({}^{-1}\) EDTA; and, 10 mmol L\({}^{-1}\) MgCl\({}_{2}\)), 0.75 mL of reaction substrate, 0.15 mL of 1.6 mmol L\({}^{-1}\) NADH, 0.1 mL of ADH enzyme extract (1.5 mg/3 mL phosphate buffer, pH = 8.6), and 0.5 mL of crude enzyme supernatant, and determined at a reaction temperature of 30 \({}^{\circ}\)C and wavelength of 340 nm. Sodium hydroperoxide linoleate was used as the substrate for determination of enzyme activity, and the reaction substrate comprised 10 mL of distilled water, 200 \(\mu\)L of 10 mmol L\({}^{-1}\) sodium linoleate, 400 \(\mu\)L of LOX enzyme extract (1 mg/10 mL phosphate buffer, pH = 9.0) that was heated in a water bath for 2 h at 30 \({}^{\circ}\)C. Change in absorbance per min at OD\({}_{340}\) was recorded 15 s after addition of the enzyme solution, where each change in OD value was denoted as 1 U [32]. Enzyme activity was measured three times per sample.  
The method of extraction of ADH enzymes was similar to that of LOX enzymes, where the reaction system comprised 0.15 mM NADH, 80 mM acetaldehyde, and enzyme extract. Changes in absorbance per min at OD\({}_{340}\) were recorded.  
AAT acyltransferase activity was determined using a slightly modified version of the method described by [PERSON] et al. [25], where 3.0 g samples of fruit were ground in 0.1 g PVPP using a pre-chilled mortar and pestle, homogenized in 6.0 mL of 0.1 mol/L potassium phosphate buffer (pH = 7.0), and centrifuged at 12,000\(\times\)\(g\) (4 \({}^{\circ}\)C) for 30 min. The reaction system, which comprised 0.5 mol\(\cdot\)L\({}^{-1}\) Tris-HCl buffer (pH = 7.0), 11.6 mmol\(\cdot\)L\({}^{-1}\) MgCl\({}_{2}\), 0.3 mmol\(\cdot\)L\({}^{-1}\) acetyl-CoA, 10 mmol\(\cdot\)L\({}^{-1}\) butyl alcohol, and 0.6 mL of supernatant, was maintained at 35 \({}^{\circ}\)C for 15 min, before 150 \(\mu\)L of 20 mmol\(\cdot\)L\({}^{-1}\) dithio-bis-nitrobenzoic acid was added and kept at room temperature for 10 min. Then, AAT activity was measured at 412 nm 15 s after the enzyme solution had been added; changes in absorbance per 1 min at OD\({}_{412}\) were recorded, where each change in OD value was denoted as 1 U ([PERSON] et al., 1981).