Practicum 5

Enzyme Activity and Characterization of Plant Pigments

The seeds were germinated can be a source of enzymes of plant tissues, although the enzymes
obtained in the form of crude enzymes. For the purposes of the experiment simple enzyme,
germ extract can be used.
A. Enzyme activity:
A.1 The effect of temperature on the enzyme activity of amylase (enzyme activity
assay qualitatively)
Tools and materials:
1.
2.
3.
4.
5.
6.
7.

Starch solution
I2KI solution.
The enzyme amylase:
Buffer solution (buffer) pH 5.6 phosphate.
6 pieces of the reaction tube.
3 fruit plate with pipetnya drops.
3 pieces of bath water temperature is different.

How to trial:
1. Prepare a water bath temperature is 450 C, room temperature and filled ice.
2. Prepare three pieces of test tubes filled as much as 2-5 mL enzyme solution and each
store at a different temperature water bath.
3. Prepare also 3 test tubes filled with 2-5 mL of starch with 2-5 mL of phosphate buffer
pH 5.6, hereinafter each put on 3 bath above.
4. Let the test tube in a water bath for a few minutes until the same temperature as the
water in the bath. If the temperature is the same, enter 1 mL of enzyme solution starch
solution into the tube and mix.
5. Perform testing for each tube with a drop lar I2 KI in the plate drops.
What can you conclude from this experiment?
A.2 Test amylase enzyme activity quantitatively (method of Bernfeld, 1955).
1. Enzyme extract is derived from mung bean sprouts, red bean sprouts and soy bean
sprouts.
2. Prepare 7 pieces reaction tubes (numbered 1-7). Amylase activity test was carried out

by mixing 0.5 mL of enzyme extract; 0.5 mL of 0.05 M sodium phosphate buffer pH

7.0 and 1.0 mL of 1% starch.
a. On the tube 1, 3 and 5, the third mixture solution was incubated for 15 min at
room temperature, then the reaction was stopped by addition of 3.5 mL of 1 N
HCl.
b. In the reaction tube 2, 4 and 6, before the enzyme extract incorporated, the
addition of HCl.
c. Reaction tube 7 is used as a blank, by mixing 0.5 mL of enzyme extract; 0.5
mL of 0.05 M sodium phosphate buffer pH 7.0 and 1 mL H2O instead of
starch.
3. Detection of starch is done by adding 0.5 mL of a solution of iodine. The absorbance
of samples was then measured at a wavelength of 580 nm.
If X is the amount of starch in the beginning of the experiment and Y is the number of
remaining starch, the amount of starch that decomposes during the experiment (Z) is the
XY and the percentage of biodegradable starch which can be calculated by using the
formula (Z / X) * 100%.
Table 1. Enzyme activity assay
Treatment
Number

Ektstrak enzyme

Absorbance
(A)

Green beans

= Y1

Green beans

= X1

Red beans

= Y2

Red beans

= X2

Soybeans

= Y3

Soybeans

= X3

Blank

B. Characterization of plant pigments

X-Y = Z

Z1

Z2

Z3

% Starch
Lost (100 * Z
/ X)

In addition to the group of primary metabolites as components of cells and tissues of plants,
there are a group of compounds / secondary metabolites. Unlike primary metabolites,
secondary metabolites are not always found in plants, only certain plants that accumulate
secondary metabolites. Secondary metabolites are grouped into three, namely alkaloids,
terpenoids and phenolic compounds. Some secondary metabolites function as plant pigments,
including chlorophyll, carotenoids, and anthocyanins. Test the presence of plant pigments and
characterization

can

be

performed

by

thin

layer

chromatography

analysis

and

spectrophotometer
B.1. Characterization of pigments in the leaves and flowers with a spectrophotometer
Extraction
1. Provide spinach leaves and purple colored flowers, red and yellow
2. Weigh these materials each 1 g
3. Each material was crushed with a mortar
4. Scour immersed in 5 mL of 96% alcohol for 30 minutes
5. Add alcohol to a volume of 10 mL
6. Filter the extract with a glass funnel (which has been capped cotton on his
neck) to obtain an alcohol extract of leaves and flowers
Analysis with a spectrophotometer
a. Measure the absorbance of the extracts that had been prepared earlier on the
wavelength () 400-700 nm with a UV / VIS Spectrophotometer
Absorbance at
Extract

400

425

450

475

500

525

550

575

600

625

650

Green leaf
Purple
flowers
Red flower
Yellow
flowers
b. Plot the results of earlier measurements on graph paper l, in order to obtain the
absorption spectrum
c. Determine the maximum absorbance of each extract (the pigment solution)

675

700

B.2 characterization of pigments in plant tissue by Thin Layer Chromatography (TLC)

B.2.1 Analysis by TLC paper leaf extract
Extraction of leaf
1. Weigh green spinach leaves as much as 1 g, crushed with a mortar
2. Soak scours in 5 mL of 96% alcohol for 30 minutes
3. Strain the solution with a glass funnel in order to get the alcoholic extract of leaves
Analysis by TLC paper
1. Prepare Whatman filter paper No. 1. The size of 3 x 10 cm
2. Dip the filter paper tsb.pada extracted earlier, which had been placed in a vessel
3. The vessel lid, let the vine extract filter paper up to 2 cm from the top edge
4. Take the paper with tweezers, then dry by way of cooling it winds
5. Observe pigment separation occurs, describe kromatogramnya!
(Cl A: blue-green; kl b: Blue-yellow; xantofil: yellow; anthocyanin: violet ~ red)
B.2.2 Analysis carrot extract by TLC Silica gel
Extraction carrots
1. Weigh 3 sliced carrots, crushed with a mortar and soak materials scour in 5 mL of
acetone for 30 minutes
2. Strain the marinade with a glass funnel thus obtained acetone extract carrots
3. The extract is then partitioned in a test tube by adding diethyl ether: H2O (1: 1) to
obtain a layer of water and diethyl ether
Analysis by TLC Silica gel
1. Prepare a silica gel plate 3 x 7 cm
2. Apply the extract with a glass capillary gel siliga plate at a distance of 1 cm from the
bottom edge
3. Dip the TLC plate on a vessel that had been filled with a solution developer (hexane:
ethyl acetate / 7: 3)
4. The vessel lid, let the developer solution creeping up to 1 cm from the top edge
5. Remove and immediately remove TSB TLC plate, and mark the developer of high
mileage

6. Dry the plate in a way diangin-wind

7. Observe spots formed
8. Draw kromatogramnya! and specify the pigments of the integral (carotene: yellow ~
orange) and santofil (yellow)