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PRACTICAL 5

AMYLASE ACTIVITY IN GERMINATING BARLEY

INTRODUCTION

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Thousand of chemical reactions occur each instant throughout the body; this coordinated process of chemical change is named metabolism.

Metabolism includes the synthesis and breakdown of organic molecules required for cell structure and function and the release of chemical energy used for cell functions.(Vander, Sherman & Luciano, 001)

The bodys organic molecule undergo continuous transformation as some molecules are broken down while others of the same type are being synthesized.

Germination is the resumption of growth of the embryo plant inside the seed.

Seeds remain dormant or inactive until conditions are right for germination. All seeds need water, oxygen, and proper temperature in order to germinate, some seeds require proper light also. Some germinate better in full light while others require darkness to germinate.

When a seed is exposed to the proper conditions, water and oxygen are taken in through the seed coat. The embryos cells start to enlarge. Then the seed coat breaks open ( and also triggers metabolic changes in the embryo) and a radicle, the embryonic root emerges first, followed by the shoot or plumule that contains the leaves and stem.

In a germinating barley seed, food reserves stored in the endosperm, (as glucose polymer starch) and during seed germination, the starch is hydrolysed and mobilized by enzyme amylase into maltose.

This enzyme hydrolyses the a (1-4) glycosidic bonds between pairs of glucose units to release the disaccharide maltose. Maltose is then hydrolysed to single glucose molecules by a second enzyme called glycosidase. Glucose can enter the glycolytic pathway where it is utilized for producing ATP and carbon molecules for biosynthesis.( Campbell & Reece, 00), when the starch reserves are exhausted, the seedling has usually reached a stage where energy and carbon requirements can be met by photosynthesis.

Photosynthesis is the process by which plants make food, is the process by which sugars and more complex compounds are made from carbon dioxide and water in cells containing chloroplasts. The chemical energy is produced from solar energy in the presence of the pigment chlorophyll.

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The aim of this exercise is to determine the comparative amylase activity in the dormant barley seeds, germinating barley seeds and barley seedlings by measuring their rates of starch hydrolysis, and to interpret this results in terms of the metabolic role of amylase during plant development.

MATERIALS AND METHODS

For the preparation of amylase extract from germinating barley, 10 grains were crushed with mortar and pestle to a fine paste, and 10ml of buffer was added, to extract the amylase into solution. The extract was filtered into a 100ml beaker, and the volume was recorded.

A five-fold dilution of the filtered extract was made by adding 5 ml to the measuring cylinder and buffer was added to make the total volume 5ml.

The control extract was prepared by adding 5ml of the diluted filtered extract to a test tube and place it in the boiling water bath for 10minutes, after that period of time it was removed from the boiling water bath and leave it to cool at room temperature.

And for the 10 dormant barley seeds and 10 whole barley seedlings the same method was used, including the use of boiled controls.

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

The rate of starch hydrolysis can be determined by making up a reaction mixture containing starch in a buffer of appropriate pH. Amylase extract is added, and the samples are tested with starch indicator (iodine) it gives a blue- black colour when bound to starch, and the samples are tested at constant time intervals of 1 minute

RESULTS

Table 1 represents a description of the amylase activity of developmental stages of barley, and time taken to reach achromic point.

NUMBER OF GRAINSTYPES OF BARLEY SEEDSFILTRATE VOLUMETIME

AMYLASE ACTIVITY LEVEL

10GERMINATING BARLEY8.5ML MINUTES Quite High

10DORMANT BARLEY8MLNO COLOUR CHANGE No reaction

10WHOLE BARLEY SD.ML1 MIN.Quite high

Among these developmental stages of barley, the amylase activity was highest in the germinating barley, followed by whole barley seedling, with dormant barley seeds showing not activity at all ( Table 1).

Calculation of amylase activity in the germinating barley

Concentration of the starch= 0.5% = 500mg/100ml = 5mg/ml

i) So, for 1ml of solution = 5mg/ml x 1ml = 5mg of starch

time taken to reach the achromic point for the germinating barley = minutes

ii)average amount of starch hydrolysed/min to reach the achromic point =5mg = 0.55mg/min

min

iii) give ten grains were used to make the extract, number of grains per ml

= number of grains =10grains = 1. grain/ml

number of ml extract8.5ml

= 1. grain/ml = 0. grains/ml x 1ml = 0. grains

5ml

So, mg starch hydrolysed/min/barley grain

= number of mg/ minute hydrolysed= 0.55mg/min = .75 mg/min/grains

number of grains 0.grains

Amylase activity = .75 mg/minute/grain for germinating barley.

For the amylase activity for the dormant and whole seedling refer to the table

Table represents amylase activity for different types of seeds

TYPES OF BARLEY SEEDSAmylase activity ( mg/min/grain)

Germinating seed.75

Dormant seed0

Whole seedling

In this table we are just reinforcing what was stated in table 1- but here we can key in number by saying -

Amylase activity was highest in the germinating experimental treatment, which had an activity of .75 mg/min/grain followed by the whole seedling experimental which had an activity of mg/min/grain and followed by the dormant seeds which did not show any amylase activity at all.

Table represents reaction of different mixtures with Benedicts reagent to determine maltose

Reaction mixtureresults

4 a) reaction mixture from (d)Red-yellow, greenish colour

4 b) controlBright blue color

Table indicates that cuprous oxide precipitate was present in the reaction tube containing amylase extract (from part d), as indicated by its resulting red-yellow, greenish colour.

Discussion

In terms of the metabolic rate of amylase during barley plant development, in the germinating barley seed , a lot of energy (ATP) is needed for cellular respiration, and for the seedling to germinate, so a lot of amylase is required in order to breakdown starch.

The amylase is able to breakdown starch into maltose, but unable to breakdown the cellulose in the cell wall into smaller molecules, despite both being polymers of glucose because as amylase is an enzyme it is very specific as to type of bonds it breaks down

In the dormant seed very little/ no amylase is produced, for the reason that as the seed is not growing or developing, very little energy is needed in order to respire, therefore minimal amylase is produced, to preserve resources and energy for germination, consequently very little starch is broken down, there is why the iodine remains blue black colour and no change of colour was observed.

Furthermore, the control treatments for each stage did not show any amylase activity. This was because the enzyme was denatured after boiling and was not able to break down starch. This shows that amylase was in fact the active agent responsible for hydrolyzing starch, as if it were not starch would have still been broken down after its boiling.

Identifying maltose as the product of starch hydrolysis by amylase

Maltose was produced as the Benedicts reagent reacted with maltose with the mixture to form a cuprous oxide precipitate( as was expected in the experiment it confirms that the amylase enzyme does catalyse the reaction of starch) from maltose in germinating barley seed. In the 4b) the control tube it only confirms that the amylase is responsible for the production of maltose.

During this practical a number of errors may have been made, which could have to some extent altered the results obtained. These mey include measurement errors, improper grinding of the grains, improper cleaning of the equipment between testing the different stages of seed development leading to contamination and even calculation error.

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