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
degrade

producing

genes
(for enzyme synthesis)

increasing

parts of fruit

resulting in

(chlorophyll, acids, starches, pectin, proteins, etc.)

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

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

METHODS (continued):
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

1 2 3 C 4

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.

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

2
3
DAY 1

C 4

2 3
DAY 3

1 C 4 5

1 2 C 3
DAY 5

5
4

2 3
DAY 7

5 C 4

DATA (continued):
(1) Banana

(2) Kiwi
(3) Mango

(5) Pineapple
(C) Water (control) (4) Papaya

DAY 1
diameter of wells (mm)
Day 1 3 (C) Water 8.5 8.7 (1) Banana 8.8 9.0 (2) Kiwi 12.5 13.1 (3) Mango 10.1 9.8 (4) Papaya 9.2 9.5 (5) Pineapple 12.8 13.0

5
7

8.5
8.3

10.0
9.7

13.5
14.4

11.4
13.0

11.2
12.5

15.5
16.0

DATA (continued):
Ripening & Protease Activity
18 16 14

Diameter (mm)

12 10 8 6 4 2 0 1 3 Days 5 7

Water Banana Kiwi Mango Papaya Pineapple

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
degrade

producing

genes
(for enzyme synthesis)

increasing

parts of fruit

resulting in

(chlorophyll, acids, starches, pectin, proteins, etc.)

Explanation (continued) - REVISED MODEL

wound/pests/pathogens methionine
reacts with

temperature increase

produces

oxygen

to be converted to

H2C=CH2
(ethylene)

inhibits growth of/diffuses into

induces/turns on

enzymes
catalyze

produce

genes
(for enzyme synthesis)

biochemical reactions
degrade

Amylase: starch --> sugar Pectinase: pectin --> less pectin Kinase: acids --> neutral Protease: proteins --> amino acids

increases

fruit tissues

results in

(chlorophyll, acids, starches, pectin, proteins, etc.)