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Amylase Activity in Germinating Barley

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Biology Practical 3

Title:

Amylase activity in germinating barley

Introduction:

A process that occurs in order to maintain life in living things is called metabolism. Metabolism is a set of chemical reactions that helps to allow organisms to reproduce and grow, help to maintain their structures and also to respond to their environments. Triglycerides is the energy that is required for the de novo synthesis of proteins during the first stages of germination. Fatty acids of the oleosomes are released and move into glyoxysomes upon water uptake, which is used to produce ATP (Brandt, 2002). The hypothesis for this experiment is that the higher the amylase activity, the higher the rate of seed germination. The aim for the experiment is to investigate metabolic processes in a living organism by extracting an active enzyme and using it to catalyse a specific biochemical reaction.


Methods:

Preparation of amylase extract from germinating barley

10 germinating barley seeds were patted with paper towel and weighed. The total mass was recorded. The 10 germinants were crushed with pestle and mortar. Crushing was continued up to 3 minutes while 10 ml of buffer was slowly added. The solution was filtered using a tea strainer into a 100 ml beaker. The amylase extract was poured into a measuring cylinder, and volume was recorded. Returned solution to beaker. 20 ml of buffer was auto-pipetted into a measuring cylinder and then 5 ml of the amylase extract was added. A control extract was prepared by adding 5 ml of the diluted amylase extract into a test tube and placed in a boiling water bath for 10 minutes. Removed after 10 minutes and left to cool.

Determination of amylase activity in germinating barley by measuring the rate of starch hydrolysis

One drop of iodine was placed into each 21 labelled wells. 5 ml of buffer and 1 ml of 0.5% starch solution was added to a test tube (reaction mixture). Added a single drop of the reaction mixture to a drop of the iodine in the well labelled T (test) using a pasteur pipette. Remixed the diluted amylase extract and then add 1 ml diluted amylase extract to the reaction mixture in the test tube (time= 0 minutes). Starting with well number 0, immediately, after 1 minute intervals, added a drop of the amylase reaction to sequential wells until the achromic point was reached. Repeated experiment using identical volumes of buffer and starch, but replace the diluted amylase extract with the same volume of boiled control extract. Calculated the activity of amylase in the germinating barley.

Identifying maltose as the product of starch hydrolysis by amylase

Using the saved reaction tube for maltose determination, added 2 ml of this amylase reaction mixture to a test tube containing 2 ml of Benedict’s reagent. Prepared a control by adding 5 ml of buffer to 1 ml of 0.5% starch solution in a clean test tube. Mixed it, then transfer 2 ml of this into a new test tube containing 2 ml of Benedict’s reagent. Placed two Benedict’s reagent tubes in the boiling water bath for 10 minutes, then examined for presence of cuprous oxide precipitate.

Presence of amylase activity during barley development

Prepared extracts of 10 dormant barley seeds and 10 whole barley seedlings. Same procedure as the first part of the experiment.  Determined the amylase activity in these extracts.

Sample calculation for germinating barley:-

0.77g (Volume of barley seeds) / 8 ml (filtered amylase extract) = 0.096 g/ml

Amylase extract was diluted further to a factor 1.5, 10 ml amylase extract and 15ml buffer, to a total of 25ml

        0.096 g/ml x 10ml = 0.96g

0.96(g) / 25 (ml) = 0.038 g/ml of diluted amylase extract

It took 15 minutes for the amylase mixture reaction to reach the achromic point, so the amylase reaction rate is:

5mg/15min/0.038 g/ml = 8.772 mg/min/g

(5mg from the equation is the starch hydrolysed)

Results:

Table 1: Amylase activity and maltose activity in dormant, germinating and whole seeds

Dormant

Germinating

Whole

Control (Bo:1)

Treatment

Control

Treatment

Control

Treatment

Amylase

No change > 20 min

No change > 20 min

No change > 20 min

11 min

No change > 20 min

15 min

Maltose

No change (-)

No change (-)

No change (-)

Slight brick red (+)

No change (-)

Slight brick red (+)

Weight

0.38

0.38

0.77

0.77

0.40

0.40

Volume after filtration

7.5

7.5

8.0

8.0

8.2

8.2

...

...

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