1. Enzymatic Isolation of Tomato Cuticles
Place several commercial or cultivated tomatoes in a bowl. Peel the skin from the fruits in large sections; discard the inner pericarp tissue. Wash the tomato skins with deionized water and preserve them under water in a beaker.
Prepare a 50 mM pH 4.0 sodium acetate buffer (at 31 °C) by putting 1.22 g of sodium acetate trihydrate (Mr. 136.08 g/mol) and 2.34 ml glacial acetic acid (17.485 M) in a beaker, adding 200 ml of deionized water, and then adjusting the pH to 4.0 at 31 °C. Prepare a mixture containing 4 ml of pectinase (EC 3.2.1.15; 10 U ml-1, TCI America), 0.2 g of cellulase (EC 232.734.4; 1.3 units/mg solid, Sigma Aldrich), and 13 mg NaN3, and then add 196 ml of sodium acetate buffer to the enzyme mixture to obtain 200 ml of the final enzyme cocktail.5 Completely immerse the peeled tomato skin in the enzyme cocktail and incubate at 31 °C for 24 hours with constant shaking (G24 Environmental Incubator Shaker; New Brunswick Scientific Co.).
Collect the tomato skins using a kitchen strainer or Büchner funnel and wash them with deionized water. Thereafter, place them in a vacuum oven at room temperature for an hour. Preserve the dried tomato skins in a labeled and capped bottle for subsequent dewaxing procedures.
1. Enzymatic Isolation of Tomato CuticlesPlace several commercial or cultivated tomatoes in a bowl. Peel the skin from the fruits in large sections; discard the inner pericarp tissue. Wash the tomato skins with deionized water and preserve them under water in a beaker.Prepare a 50 mM pH 4.0 sodium acetate buffer (at 31 °C) by putting 1.22 g of sodium acetate trihydrate (Mr. 136.08 g/mol) and 2.34 ml glacial acetic acid (17.485 M) in a beaker, adding 200 ml of deionized water, and then adjusting the pH to 4.0 at 31 °C. Prepare a mixture containing 4 ml of pectinase (EC 3.2.1.15; 10 U ml-1, TCI America), 0.2 g of cellulase (EC 232.734.4; 1.3 units/mg solid, Sigma Aldrich), and 13 mg NaN3, and then add 196 ml of sodium acetate buffer to the enzyme mixture to obtain 200 ml of the final enzyme cocktail.5 Completely immerse the peeled tomato skin in the enzyme cocktail and incubate at 31 °C for 24 hours with constant shaking (G24 Environmental Incubator Shaker; New Brunswick Scientific Co.).Collect the tomato skins using a kitchen strainer or Büchner funnel and wash them with deionized water. Thereafter, place them in a vacuum oven at room temperature for an hour. Preserve the dried tomato skins in a labeled and capped bottle for subsequent dewaxing procedures.
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