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Aissvarya Subramaniyam 28693019 PRACTICAL 3: AMYLASE ACTIVITY IN GERMINATING BARLEY Introduction Metabolism refers to the totality of an organism’s chemical reactions to maintain life (Campbell et al. 2015). Enzymes are crucial to metabolism, acting as a catalyst to drive desirable reactions requiring energy that will not occur by them. During seed germination, amylase is activated, which hydrolyses starch and converts it to maltose, which is further broken down to glucose by another enzyme, glucosidase. The aim of this experiment is to investigate the biochemical reaction of barley by the extraction of amylase enzyme, and hence, investigating the amylase activity and the presence of maltose at different life stages of barley. The hypothesis is that amylase activity is highest in germinating (3-day) seed and lowest in dormant (0-day) seed and Benedict’s reagent is hypothesized to show a positive red-yellow precipitate in the presence of maltose in both germinating and whole seedling, but a negative result in dormant seeds. Methods Ten dormant seeds were dried, weighed and crushed. Ten ml of buffer was slowly added to the paste. The solution was filtered and the volume recorded. A diluted amylase extract was made by a five-fold dilution. Five ml of control extract was placed in boiling water bath for 10 minutes to determine the effect of high temperature on the activity of amylase to hydrolyze starch since excessive heat leads to enzyme denaturation. A drop of iodine was placed into 21 labelled wells. A drop of the reaction mixture made from 5 ml of buffer, 1 ml of 0.5% starch solution and 1 ml of diluted amylase extract was immediately added to the well labelled 0 and to sequential wells every 1 minute intervals until reaching the a chromic point. This was repeated with the boiled control. Measuring the amount of starch hydrolysed/min to reach the achromic point, and finding the mass of barley tissue added calculated amylase activity. It was calculated as mg starch hydrolysed/min/g of barley tissue.

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