Research Question How will the addition of different pH buffers to amylase affect the rate of starch digestion measured using starch and iodine?
Introduction Amylase is an enzyme found in human saliva and pancreas. It is the digestive enzyme that is needed to breakdown starch molecules. Amylase must be kept at certain conditions to function at its optimum level. This experiment will explore the effect of pH (1, 4, 7, 10, and 14) on the function of amylase by using starch and iodine.
Usually iodine has a orange-yellow color, but iodine and starch react to produce a dark blueblack color, so iodine may be used as an indicator to show the rate at which starch is broken down.1 This occurs when polyiodide chains are formed from starch and iodine. However, as starch is hydrolyzed into smaller carbohydrate units, the blue-black color does not appear. Therefore, using this iodine test, the effects of pH on the function of amylase can be determined by the time it takes (if at all) for the iodine to remain its orange-yellow color.2
Hypothesis Enzymes must be kept at certain conditions to function at its optimum level. Indeed, factors that may cause the enzyme to denature are: pH, temperature, and salt concentrations. When an enzyme is denatured, it can no longer bind to the active site, and therefore cannot carry out its functions. Therefore, adding pH buffer to amylase will affect the enzyme’s function upon its addition to starch, which can be indicated by the iodine test. In fact, if the enzyme is denatured by the pH buffer, the iodine will turn blue-black when starch and enzyme solutions are added because the enzyme didn’t digest the starch. However, if the optimum pH is added 1
" H o w A m y l a s e Wo r k s . " L e s s o n S n i p s . L e s s o n S n i p s , n . d . We b . 1 0 J a n 2 0 1 1 . < h t t p : / / w w w. l e s s o n s n i p s . c o m / d o c s / p d f / a m y l a s e w o r k . p d f > . 2 Senese, Fred. "How does starch indicate iodine?."General Chemistry Online. General C h e m i s t r y O n l i n e , n . d . We b . 1 0 J a n 2 0 1 1 . .
to amylase, the iodine will slowly turn orange-yellow because the enzyme will digest the starch.
The optimum pH level for amylase depends on the type of amylase. For example, the optimum pH level for alpha-amylase is is 6.7 to 7, gamma-amylase is 3.3 Therefore, from the pH buffers that will be used in this experiment, pH 7 or pH 4 will show optimum activity. Then, the extremely acidic and extremely basic buffers will probably denature the amylase.
" E f f e c t s o f p H . " Wo r t h i n g t o n B i o c h e m i c a l C o r p o r a t i o n s ( 2 0 1 1 ) : n . p a g . We b . 1 0 J a n 2 0 1 1 . < h t t p : / / w w w. w o r t h i n g t o n - b i o c h e m . c o m / i n t r o B i o c h e m / e f f e c t s p H . h t m l > .
Variables Variable Measured Method of Measuring The change in iodine color with the addition of starch and enzyme solution will indicate starch hydrolysis. If the iodine turns blueblack color, it indicates the presence of starch, therefore the denatured enzyme If the iodine remains orange-yellow color, it indicates the absence of starch, therefore the proper function of amylase This mixture will be manipulated by the addition of pH buffer into diluted amylase enzyme. The pH buffers that will be used are: 1, 4, 7, 10 and 14. This mixture will then be incubated for 24 hours at room temperature 50 μl of iodine will be added into each well of the microplate 5 ml of starch solution and 500 μl of enzyme solution (amylase + pH buffer) are first mixed. 500 μl of this mixture is added into each well filled with iodine. The experiment was all carried out at room temperature (25°C) The starch and enzyme mixture were added at every 30 second interval to observe the color change of iodine. The time was kept with a stopwatch.
Rate of starch hydrolysis
Solution of amylase and pH buffer
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