1. Janet Byun EDTEP 586 December 8, 2005 Fruit Ripening and Protease Activity

2. BACKGROUND RESEARCH:  Fruit Ripening Process  Enzymes  Proteases QUESTION: How does protease activity change as fruits ripen?

3.  Are proteins that catalyze reactions in metabolism.  Bind substrates at active sites, form ES-complex.  Not consumed or altered by reactions.  Found in raw fruits and vegetables. Proteases…  Are enzymes that hydrolyze peptide bonds of proteins.  Banana, mango, papaya contain a protease called papain; kiwi contains actinidin; pineapple contains bromelin. These proteases are known to break down proteins in ripening tissues. Fruit Ripening Process…  Irreversible biochemical process between late maturation, early senescence.  Changes in color, texture, aroma, flavor.  Initiated by ethylene: o A gaseous plant hormone produced by the fruit. o Production rate increases with ripening. o Induces/regulates genes that encode for enzymes. Enzymes…

4. HYPOTHESIS : Riper fruits have increased protease activity compared to less ripe/unripe fruits. INITIAL MODEL: produces H 2 C=CH 2 (ethylene) induces/turns on genes enzymes (for enzyme synthesis) producing degrade parts of fruit resulting in increasing (chlorophyll, acids, starches, pectin, proteins, etc.) *Theoretical = black text in white boxes

5. METHODS:  Prepared four petri dishes with 5mm thickness of gelatin each.  Created five 8.5mm width wells.  Obtained 20 fruits with proteases known to degrade protein in gelatin. o 4 bananas o 4 kiwis o 4 mangos o 4 papayas o 4 pineapples

6. METHODS (continued):  DAY 1: Collected fresh fruit juice from 1-day old fruits of each type of fruit.

7.  Placed 5 drops of each type of fresh fruit juice into assigned, numbered wells. o C = water (for control) o 1 = banana o 2 = kiwi o 3 = mango o 4 = papaya o 5 = pineapple  Petri dishes with filled wells then sat for 5 hours at room temperature. Diameters of wells measured and recorded.  Procedure repeated for 3-day old, 5-day old, and 7-day old fruits. METHODS (continued): 5 1 2 C 3 4

8. ASSUMPTIONS:  Ethylene source purely from fruit; not from surroundings.  Temperature and ventilation of environment for fruits constant.  No wounds, pests, or pathogens affecting fruits.  Breakdown of gelatin strictly due to proteases in fruits.  Proteases active under room temperature.  7 days would be a sufficient amount of time to collect data for ripening.

9. DAY 3 DAY 5 DAY 1 DAY 7 DATA: 5 1 2 C 3 4 5 1 2 C 3 4 5 1 2 C 3 4 5 1 2 C 34  C = Water  1 = Banana  2 = Kiwi  3 = Mango  4 = Papaya  5 = Pineapple

10. Day(C) Water(1) Banana(2) Kiwi(3) Mango(4) Papaya(5) Pineapple 18.58.812.510.19.212.8 38.79.013.19.89.513.0 58.510.013.511.411.215.5 78.39.714.413.012.516.0 DATA (continued): diameter of wells (mm) DAY 1 (1) Banana (5) Pineapple (C) Water (control) (4) Papaya (3) Mango (2) Kiwi

11. Riper fruits had increasing protease activity present compared to less ripe fruits. DATA (continued):

12. CULMINATING ARGUMENT: Claim - Ripe fruits have an increase in protease activity compared to less ripe/unripe fruits because as fruits ripen, they produce increasing amounts of ethylene, signaling more enzyme genes to be translated and transcribed, and thus more enzymes to be produced to hydrolyze proteins. Explanation - Data collected and represented in my graph is evidence supporting an increasing trend in protease activity as fruits ripen.

13. HYPOTHESIS : Riper fruits have increased protease activity compared to less ripe/unripe fruits. INITIAL MODEL: produces H 2 C=CH 2 (ethylene) induces/turns on genes enzymes (for enzyme synthesis) producing degrade parts of fruit resulting in increasing (chlorophyll, acids, starches, pectin, proteins, etc.) *Theoretical = black text in white boxes

14. wound/pests/pathogens produces H 2 C=CH 2 (ethylene) induces/turns on genes temperature increase (for enzyme synthesis) produce degrade fruit tissues results in increases (chlorophyll, acids, starches, pectin, proteins, etc.) enzymes oxygen reacts with methionine to be converted to inhibits growth of/diffuses into catalyze biochemical reactions Explanation (continued) - REVISED MODEL Amylase: starch --> sugar Pectinase: pectin --> less pectin Kinase: acids --> neutral Protease: proteins --> amino acids