Fruit ripening and protease activity

Fruit Ripening and Protease Activity Janet Byun EDTEP 586 December 8, 2005 QUESTION: How does protease activity change as fruits ripen? BACKGROUND RESEARCH: Fruit Ripening Process Enzymes Proteases 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… 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 HYPOTHESIS: Riper fruits have increased protease activity compared to less ripe/unripe fruits INITIAL MODEL: *Theoretical = black text in white boxes produces H2C=CH2 (ethylene) induces/turns on enzymes producing genes (for enzyme synthesis) degrade parts of fruit resulting in (chlorophyll, acids, starches, pectin, proteins, etc.) increasing 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 bananas o kiwis o mangos o papayas o pineapples METHODS (continued): DAY 1: Collected fresh fruit juice from 1-day old fruits of each type of fruit METHODS (continued): Placed drops of each type of fresh fruit juice into assigned, numbered wells o C = water (for control) o = banana o = kiwi o = mango o = papaya o = pineapple C Petri dishes with filled wells then sat for hours at room temperature Diameters of wells measured and recorded Procedure repeated for 3-day old, 5-day old, and 7-day old fruits 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 DATA: C = Water 1 = Banana 2 = Kiwi 3 = Mango 4 = Papaya 5 = Pineapple C 3 DAY C DAY 1 C DAY C DAY DATA (continued): (1) Banana (2) Kiwi (5) Pineapple (C) Water (control) (3) Mango (4) Papaya DAY diameter of wells (mm) Day (C) Water (1) Banana (2) Kiwi (3) Mango (4) Papaya (5) Pineapple 8.5 8.8 12.5 10.1 9.2 12.8 8.7 9.0 13.1 9.8 9.5 13.0 8.5 10.0 13.5 11.4 11.2 15.5 8.3 9.7 14.4 13.0 12.5 16.0 DATA (continued): Ripening & Protease Activity 18 16 14 Water Banana Kiwi Mango Papaya Pineapple 12 10 Diameter (mm) Days Riper fruits had increasing protease activity present compared to less ripe fruits 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 HYPOTHESIS: Riper fruits have increased protease activity compared to less ripe/unripe fruits INITIAL MODEL: *Theoretical = black text in white boxes produces H2C=CH2 (ethylene) induces/turns on enzymes producing genes (for enzyme synthesis) degrade parts of fruit resulting in (chlorophyll, acids, starches, pectin, proteins, etc.) increasing Explanation (continued) - REVISED MODEL wound/pests/pathogens temperature increase methionine reacts with oxygen produces to be converted to H2C=CH2 (ethylene) inhibits growth of/diffuses into induces/turns on enzymes produce (for enzyme synthesis) catalyze biochemical reactions Amylase: starch > sugar Pectinase: pectin > less pectin Kinase: acids > neutral Protease: proteins > amino acids degrade fruit tissues genes results in (chlorophyll, acids, starches, pectin, proteins, etc.) increases [...]...DATA (continued): Ripening & Protease Activity 18 16 14 Water Banana Kiwi Mango Papaya Pineapple 12 10 8 6 Diameter (mm) 4 2 0 1 3 5 7 Days Riper fruits had increasing protease activity present compared to less ripe fruits CULMINATING ARGUMENT: Claim - Ripe fruits have an increase in protease activity compared to less ripe/unripe fruits because as fruits ripen, they produce increasing... 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 HYPOTHESIS: Riper fruits have increased protease activity compared to less ripe/unripe fruits INITIAL MODEL: *Theoretical = black text in... of/diffuses into induces/turns on enzymes produce (for enzyme synthesis) catalyze biochemical reactions Amylase: starch > sugar Pectinase: pectin > less pectin Kinase: acids > neutral Protease: proteins > amino acids degrade fruit tissues genes results in (chlorophyll, acids, starches, pectin, proteins, etc.) increases ... compared to less ripe/unripe fruits INITIAL MODEL: *Theoretical = black text in white boxes produces H2C=CH2 (ethylene) induces/turns on enzymes producing genes (for enzyme synthesis) degrade parts of fruit resulting in (chlorophyll, acids, starches, pectin, proteins, etc.) increasing Explanation (continued) - REVISED MODEL wound/pests/pathogens temperature increase methionine reacts with oxygen produces ...QUESTION: How does protease activity change as fruits ripen? BACKGROUND RESEARCH: Fruit Ripening Process Enzymes Proteases Fruit Ripening Process… Irreversible biochemical... (continued): Ripening & Protease Activity 18 16 14 Water Banana Kiwi Mango Papaya Pineapple 12 10 Diameter (mm) Days Riper fruits had increasing protease activity present compared to less ripe fruits... contains bromelin These proteases are known to break down proteins in ripening tissues HYPOTHESIS: Riper fruits have increased protease activity compared to less ripe/unripe fruits INITIAL MODEL: