Authcate Prac 3 - practical3 PDF

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1. 10 germinating seeds were dried, weighted and crushed into fine paste. Further crush for 3 minutes after adding 10 ml of buffer to extract the amylase into solution. Amylase extraction were filtered and measured the volume. A five to one dilation is done with 5ml of amylase extract and 20ml of buffer is added to form diluted amylase extract. A boiled control with 5ml of diluted amylase extract is placed in water bath for 10 minutes. One equal drop of iodine was added into each 21 labelled wells on ceramic test plates. Then, 5 ml of buffer and 1 ml of 0.5% starch solution were added in test tube to form reaction mixture and one drop is added into well labelled T(for test) to familiarize with colour change. Next, 1ml of diluted amylase extract was added into reaction mixture then mix well to form amylase reaction mixture(ARM). Immediately, start added one drop of ARM into well labelled from 0 at 1-minute interval until achromic point reached. Experiment is then repeated by replacing diluted amylase extract with boiled control extract. The purpose of boiled control is used to compare with ARM and observe the differences. Note that achromic point will not reached as amylase has been denatured in high temperature thus no starch will be hydrolysed. The activity of amylase in germinating barley can be calculated by divide the average amount of starch hydrolysed per minute by the product of the mass of the barely seeds over the total volume of filtrate over the dilution factor, giving mg of starch hydrolysed/min/g of barely tissue. Benedict’s test is then conducted for determination of maltose. Two test tubes are prepared with 2ml of Benedict’s reagent , then 2ml of amylase reaction mixture was added into one of them. Then, 2ml of a mixture of 5ml of buffer and 1 ml 0.5% starch solution acts as a control was added into another test tube. Both test tubes were placed in water bath for 10 minutes and presence of cuprous oxide precipitate was examined. Repeat entire experiment with dormant seeds and seedlings.

2. CALCULATIONS:

3 days ( Germinating barley seeds ) The concentration of starch solution = 500 mg / 100 mL = 5 mg/mL The amount of starch added = 5mL = 5mg The time taken to reach the achromic point = 12.43 minutes 5 mg 12.43 min

The amount of starch hydrolysed per minute =

= 0.402 mg/min

The volume of filtered amylase extract = 8.3 mL The weight of barley seeds = 1.083g The amount of barley tissue in 5mL extract =

1.083 g ×5 mL =0.652 g 8.3 mL

The amount of barley tissue in 1mL diluted extract = The amylase activity =

0.652 g ×1 mL 25 mL

averageamount of starchhydrolysed/min mass of tissue ∈1 mL diluted extract

= 0.0261g =

0.402mg / min 0.0261 g

=

15.40mg/min/g

3.

This experiment aimed to analyse amylase activity in dormant, germinating and whole barley seedlings. The results showed that amylase activity of dormant seeds is zero since it’s inactive and embryo seed has very low metabolism. Gibberellic acid is absent since no imbibition happened therefore no amylase produced in aleurone layer. Achromic point is not available since no starch is being hydrolysed in absence of amylase. Achromic point is the time where mixture reaction can no longer initiate the colour change of iodine. The greater the amount of starch, the higher the achromic point because it requires longer time for all the starch to be fully hydrolysed. Germinating seeds have the highest amylase activity since it reached achromic point fastest. Production of gibberellic acid initiates imbibition resulting to an increasing in amylase concentration which involved in metabolizing starch into maltose in germinating stage. Most

of the starch is hydrolysed to generate ATP for growth and development. The amylase activity slows down after several days of germination where germinating seeds developed into seedlings that can undergo photosynthesis to achieve energy and carbon requirements by producing sucrose therefore starch hydrolysis process is no longer required. Decrease in amylase activity might also due to glucose complex such as cellulose that cannot be broken down by amylase. Benedict’s test is positive for both germinating seeds and barley seedlings where brick red precipitate formed indicates amylase enzyme hydrolyses α-1,4 glycosidic linkages in starch to produce maltose. Benedict’s test gave negative result in dormant seeds indicates that maltose is absent due to extremely low amylase activity. The experiment fulfilled the aim quite accurately since it allows us to acknowledge the relationship of enzyme activity’s rate and metabolism of amylase in seeds through study of achromic point and benedict’s test. Moreover, sets of control was carried out to enhance the accuracy of the result as a comparison can be made to further prove that amylase is responsible for the production of maltose. Another strength is the use of different stage of barley seed which allow the study of amylase activity and metabolic rate of various stages of barley to be more distinct and effective. We also gathered data from other groups to obtain more reliable result. Nevertheless, the use of iodine was effective as it is specific on starch only and allow a visible colour change from yellow to blue-black in presence of starch. Benedict’s reagent used is a good indicator to determine the presence of sugar such as maltose as precipitate can be easily observed. Furthermore, the amount of iodine solution added to each well was almost identical, thus the color change when amylase extract was added to the iodine solution could be detected easily, achromic point could be observed easily. However, a weakness in this experiment was the filtered amylase extract are entrapped in filtered paper during filtration causing loss of amylase and affect the results obtained. A Buchner funnel can be used instead to ensure all extract being transferred into filtrate from residue. Centrifugation can be even carried out instead of filtration to completely separate amylase extract from residue by centrifugal force. Another limitation in this experiment is taking different initial mass of seeds at different stages. To allow a more reliable and accurate result, one variable should be kept constant for a comparison therefore the mass of barley seeds in all stages should kept constant.

Nevertheless, the strength used in crushing and grinding process to extract amylase from the seeds may be improper and insufficient leading to some amylase trapped in the seeds due to human limitation in maintaining constant and appropriate strength manually to allow perfect extraction of amylase from seeds. As an improvement , a blender could be used to break down the seeds completely into paste hence lead to complete extraction of amylase from the seeds therefore increase the accuracy of the result.

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