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 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

5
C

3

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

1 = Banana
2 = Kiwi
3 = Mango
4 = Papaya
5 = Pineapple

2

5

C

3

2
3

4

DAY 1

5

C
4

DAY 3

1

1
2

5

C
3
DAY 5

1

2

C
3

4
DAY 7

5

4


DATA (continued):
(1) Banana

(2) Kiwi

(5) Pineapple
(C) Water (control)

(3) Mango

(4) Papaya
DAY 1
diameter of wells (mm)

Day

(C) Water

(1) Banana

(2) Kiwi

(3) Mango

(4) Papaya

(5) Pineapple

1

8.5

8.8

12.5

10.1

9.2

12.8

3

8.7

9.0

13.1

9.8

9.5

13.0

5

8.5

10.0

13.5

11.4

11.2

15.5

7

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
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 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