Effect Of Amylase On Starch

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Bio1022 – Practical 3

Aim: An investigation of amylase activity on the different stages of barley seeds development
Metabolism involves a series of chemical reactions which allows the organism to maintain its structure, increase its biomass, reproduce and react to their surroundings. In a dormant barley seed, starch is stored in the endosperm as a source of energy storage (King, Reiss & Roberts, 2001). Starch is subsequently broken down into its constituents, being glucose. Hence, the role of amylase within this reaction is to hydrolyze starch to maltose (Reaction 1). Lastly in order to further breakdown maltose into glucose requires another enzyme, glucosidase (Reaction 2).
Reaction 1: Starch
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After that the seeds were crushed to a fine paste with the 10ml of buffer added as the seeds were crushed, the paste is then subsequently filtered into a measuring cylinder. The solution collected after filtration is the amount of amylase extracted solution (AE), with its volume being recorded. The amylase extracted solution(5ml) is then transferred into a beaker, then a five-fold dilution of the amylase extract using 20ml of buffer. Resulting in 25ml of diluted amylase extract (DAE).
A control extract is prepared (5ml of DAE) to a test tube, which is then placed in boiling waterbath for 10minutes, after 10minutes remove the control extract and leave it to cool at room temperature.
In order to determine the amylase activity, one drop of iodine is dropped into 21 labelled wells on the ceramic test plates. A reaction mixture is prepared, 5ml of buffer and 1ml of 0.5% starch solution to a test tube. Extract one drop from the reaction mixture to the well labelled T. Turning blue-black indicating the presence of starch in the reaction mixture. Add 1 ml of diluted amylase extract to the reaction mixture. Starting with well number 0, add one drop of amylase reaction mixture to a different well each minute until the achromic point is reached (no color change). Repeat the experiment using boiled control extract instead of DAE for the determination of amylase
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As stated in the thesis “Amylase activity in dormant and germinating seeds” the amylase activity of whole seeds rises very sharply to a maximum and remains constant during the germination period. However, this discrepancy can be attributed to the age of the seeds as older seeds have significantly lower amylase concentration after long period of dormancy (Ernst, 1971). Another reason may be due to experimental errors during extraction of the amylase from the barley seeds, for example the seeds that are not grinding finely can lead to a decrease in amylase concentration.
Lastly only germinating and whole barely seeds showed the presence of maltose, indicating only within these two there is amylase present which actively hydrolyzing starch into maltose, as per reaction 1 stated above. As dormant seeds have amylase concentrations that are far too low to be detected by this type of assay, as during dormancy energy demands for this state is considered to be zero. Hence the amylase concentration in the dormant seeds are far too low to be detected in the Benedict’s reagent test (Ernst,
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