Amylase is a calcium dependent enzyme which hydrolyzes complex carbohydrates at alpha 1,4-linkages to form maltose and glucose. Amylase is an enzyme found in the germinating seeds. Imbibition process causes the release of growth plant hormone gibberelin which stimulates the synthesis of amylase. The activity of the amylase enzyme is affected by many factors such as temperature, pH, enzyme concentration, substrate concentration, and the presence of any inhibitors or activators. In germinating barley, the food reserves are stored in the endosperm. The cotyledons store food for the use of embryo in the form of starch. Amylase enzyme breaks down starch into maltose, a chain of two glucose molecules Maltose then breaks down into glucose by the enzyme glucosidase. Glucose then enters the glycolytic pathway where it is used for the production of ATP and carbon molecules for biosynthesis. Glucose is used for the growth of plumule and radicle. This process is also known as the germination process. The emergence of plumule and radicle indicate that the seeds have germinated. In germinated seeds, the blue colour of the Benedict's solution change to brick-red precipitate indicating the presence of glucose while maintaining the yellowish-brown colour of the iodine solution indicating the absence of starch. However, in non-germinated seeds, the yellowish-brown colour of the iodine solution change to blue black indicating the presence of starch while maintaining the blue colour of the Benedict's solution indicating the absence of glucose. Most of the time, when all the starch have been used up, the seedling capable of undergoing photosynthesis to produce energy and carbon.
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The higher the amylase activity, the higher the rate of seed germination. This is observed by a higher change in length of plumule and radicle. Hence, when performing the Benedict's test, the concentration of brick-red precipitate is higher seedlings and the solution remains blue for the dormant seed.
The aim of the experiment was to extract amylase from barley and to use it for the catalysis of a biochemical reaction hence investigating the amylase activity during seed germination.
Materials and methods
Ten germinating seeds were taken and using a paper towel, the germinants were patted dry and the weight of the germinating seeds were recorded. Next, using a mortar and pestle, the 10 germinating seeds were crushed into a puree. Slowly adding 10 ml of buffer, the germinating seeds were further crushed for two minutes. This will allow the amylase to go into the solution. The crushed seeds was filtered into a 100 ml beaker and the amylase extract was poured into a measuring cylinder. The volume of amylase extract was recorded. A five-fold dilution of the latter was done by pipetting 5 ml of the amylase extract and adding 20 ml of buffer to make up a total volume of 25 ml. This mixture is called the diluted amylase extract. A control was then done by adding 5 ml of the diluted amylase extract in a test tube and placing it in a water bath at 80o C for 10 minutes. When the 10 minutes have elapsed the control was removed and allow to cool to room temperature.
Next the activity of amylase per mass of germinating barley tissue is to be determined. For this, onto ceramic plates, one drop of iodine was placed into 21 wells. A reaction mixture was then prepared by adding 5 ml buffer and 1 ml of 0.5% starch solution in a test tube. Then using a pasteur pipette, one drop of the reaction mixture was removed and added to one drop of the iodine. The iodine turned blue black. This was done to ensure the presence of starch in the reaction mixture. The previously made diluted amylase extract is thoroughly remix and 1 ml of the latter was added to the reaction mixture. The mixture is called amylase reaction mixture. (As soon as the amylase reaction mixture was prepared, reaction started. Amylase started to break down starch into simple sugars). Immediately, starting with well 0 on the ceramic plate, one drop of amylase reaction mixture was added to the iodine using a pasteur pipette. At one minute interval, another drop of the amylase reaction mixture was added to another well. This was repeated until the achromic point was reached. When the achromic point had been reached, the time elapsed was recorded.
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Once the achromic point was reached, the amylase reaction mixture was kept for the determination of maltose. (Note: Benedict's reagent gives a red-yellow precipitate of cuprous oxide when boiled with maltose. This reaction does not occur with starch.) In a test tube, 2 ml of the amylase reaction mixture and 2 ml of Benedict's reagent was added. A control reaction mixture was also prepared by adding 5 ml buffer and 1 ml of 0.5% starch solution but without the amylase extract. 2 ml of the control reaction mixture was then added in a test tube along with 2 ml of Benedict's reagent. Both the Benedict's reagent tubes were placed in a water bath at 80oC for 10 minutes and then examined for presence of cuprous oxide precipitate.
All of the above steps were then repeated but with dormant seeds and seedlings. All data were then recorded for further investigation.
When preparing the amylase extract from the germinating seed, dormant seed and the seedling, the weight of the germinants and the volume of the amylase extract are given in table 1 below.
Weight of germinants (g)
Volume of the amylase extract (ml)
Table 1 6.0 ml
Table 2 below summarizes the time to reach achromic point and the test for presence of maltose for the 3 different kind of seeds.
Maltose Test (+ or -)
Referring to table 2 it is noticed that