IB Biology on Hydrolysis of starch by enzyme amylase
Research QuestionHow will the addition of different pH buffers to amylase affect the rate of starch digestionmeasured using starch and iodine?IntroductionAmylase is an enzyme found in human saliva and pancreas. It is the digestive enzyme that isneeded to breakdown starch molecules. Amylase must be kept at certain conditions tofunction at its optimum level. This experiment will explore the effect of pH (1, 4, 7, 10, and14) 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 blue-black color, so iodine may be used as an indicator to show the rate at which starch is brokendown.1 This occurs when polyiodide chains are formed from starch and iodine. However, asstarch 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 bedetermined by the time it takes (if at all) for the iodine to remain its orange-yellow color.2HypothesisEnzymes must be kept at certain conditions to function at its optimum level. Indeed, factorsthat may cause the enzyme to denature are: pH, temperature, and salt concentrations. Whenan enzyme is denatured, it can no longer bind to the active site, and therefore cannot carry outits functions. Therefore, adding pH buffer to amylase will affect the enzyme’s function uponits addition to starch, which can be indicated by the iodine test. In fact, if the enzyme isdenatured by the pH buffer, the iodine will turn blue-black when starch and enzyme solutionsare added because the enzyme didn’t digest the starch. However, if the optimum pH is added1 " 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. GeneralC 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 .<http://antoine.frostburg.edu/chem/senese/101/redox/faq/starch -as-redox-i n d i c a t o r. s h t m l > .
to amylase, the iodine will slowly turn orange-yellow because the enzyme will digest thestarch.The optimum pH level for amylase depends on the type of amylase. For example, theoptimum pH level for alpha-amylase is is 6.7 to 7, gamma-amylase is 3.3 Therefore, from thepH 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.3 " 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 n2 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 blue- black color, it indicates the presence of starch, therefore the denaturedDependent Variable Rate of starch hydrolysis 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 thatIndependent Variable Solution of amylase and pH buffer 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 Amount of iodine microplate 5 ml of starch solution and 500 μl of enzyme solution (amylase + pH buffer) are first Amount of starch and enzyme mixture mixed. 500 μl of this mixture is added into each well filled with iodine.Controlled Variables The experiment was all carried Temperature out at room temperature (25°C) The starch and enzyme mixture were added at every 30 second interval to observe Time Interval (30 seconds) the color change of iodine. The time was kept with a stopwatch. Table 1: List of variables
Apparatus and Materials pH buffer (1, 4, 7, 10, 14) Starch Amylase Iodine Hot Plate Small size beaker Medium size beaker (for serial dilution) 100 cm3 volumetric flask 1000 μl and 50 μl micropipettes Microcentrifuge tubes Vortex mixer Stopwatch Distilled waterProcedureA) Preparation of 0.1% starch 1) Mix together 0.1g of starch and 100cm3 of distilled water in a beaker 2) Place the beaker on a hot plate and stir until it becomes a homogenous solutionB) Serial dilution of amylase (10-2) 1) Add 10 μl of amylase and 990 μl of distilled water into a microcentrifuge tube Figure 1: Microcentrifuge tube4 2) Mix well with a vortex mixer Figure 2: Vortex mixer54 Image obtained from www.lightlabsusa.com/Microcentrifuge-Tubes/
C) Preparing the amylase and pH buffer solution 1) Add 50 μl of the diluted down amylase enzyme and 50 μl of the appropriate pH buffer into a microcentrifuge tube 2) Mix well with a vortex mixer 3) Incubate at room temperature for 24 hoursD) Conducting the experiment 1) Using a 50 μl micropipette, add 50 μl of iodine into each well of the microplate Figure 3: Microplate6 2) Pour 5 ml of starch and 500 μl of enzyme and pH 1 buffer mixture into a medium size beaker 3) Immediately, using the 1000 μl micropipette take out 500 μl of this mixture and extract it into the first well of iodine 4) At the same time, use the stopwatch to keep track of time 5) At every 30 second interval, add another 500 μl into the next well 6) Observe the change in color and record on a separate piece of paper 7) Repeat the steps 2) to 6) are repeated for duplicate trials for each enzyme and pH mixture (4, 7, 10, and 14)5 Image obtained from www.montreal-biotech.com6 Image obtained from http://www.abgene.com/images/products/AB-0796.jpg
Data CollectionQuantitative Data pH level 1st Trial 2nd Trial Control a) 7th interval 7th interval b) pH 1 - -b) pH 4 2nd interval 2nd interval pH 7 7th interval 7th interval b) pH 10 - -b) pH 14 *c) *c) Table 2: Change in color over time at different pH buffersa) Control group is pure amylase that has just been diluted down to 10-2b) The iodine did not turn blue-blackc) The iodine turned colorless (clear)Qualitative Data Control 1. The iodine in the well turned blue-black until the seventh 30-second interval, when it remained orange-yellow. 2. The iodine in the well turned blue-black until the seventh 30-second interval, when it remained orange-yellow pH 1 1. The iodine in the well turned blue-black continuously for both trials pH 4 1. The iodine in the well turned blue-black until the second 30-second interval, when it remained orange-yellow 2. The iodine in the well turned blue-black until the second 30-second interval, when it remained orange-yellow pH 7 1. The iodine in the well turned blue-black until the seventh 30-second interval, when it remained orange-yellow 2. The iodine in the well turned blue-black until the seventh 30-second interval, when it remained orange-yellow pH 10 1. The iodine in the well turned blue-black continuously for both trials
pH 14 1. The iodine in the well turned clear from the first interval for both trialsProcessed Data pH level 1st Trial 2nd Trial control 210 seconds 210 seconds pH 1 - - pH 4 60 seconds 60 seconds pH 7 210 seconds 210 seconds pH 10 - - pH 14 * * Table 3: Calculation of time taken for enzyme reactionRate enzyme activity = 1/time taken to hydrolyse Average time taken/s Rate = 1/Average time control 210 1/120 = 0.0083s-1 pH1 denatured denatured pH 4 60 1/60 = 0.0166s-1 pH 7 210 1/120=0.0083s-1 pH 10 denatured denatured pH 14 denatured denatured Table 4 : Rate of enzyme at different pH
Data Presentation Rate enzyme/s-1 against pH 0.018 Rate of enzyme activity 0.016 0.014 0.012 0.01 0.008 0.006 0.004 0.002 0 0 5 10 15 pHGraph 1: showing enzyme activity at different pH 250 210 210 210 200 150 Time / s 120 1st Trial 100 2nd Trial 60 60 50 0 0 0 0 0 0 0 control 1 4 7 10 14 pH Level Graph 2: Graph of time taken for enzyme reaction to occur
ConclusionThe hypothesis was supported by the results in that the amylase incubated at pH 4 and pH 7successfully hydrolyzed the starch. Even though the control (pure amylase diluted to 10-2)matched the pH 7 buffer by both hydrolyzing the starch at 210 seconds, the optimal activityoccurred with the amylase incubated at pH 4 which hydrolyzed the starch at 60 seconds.Therefore, this particular amylase that was used throughout the experiment functioned best atacidic conditions. The enzyme evidently denatured at pH buffer 1 and 10 because the iodinecontinued to turn blue-black, which means that the enzyme could not carry out its functionand starch was present. On the other hand, the amylase incubated at pH buffer 14 showed asurprising result: the orange-yellow color iodine turned into a clear substance. Ultimately, theexperiment shows that the optimal pH level for the amylase enzyme was pH 4, and the time ittook for the starch to be hydrolyzed was 60 seconds.EvaluationOverall, the experiment yielded reliable results. In fact, duplicate trials showed identicalresults for every pH buffer, which shows the accuracy of the results. Although there is nodefinite trend between the time at which the enzyme completely hydrolyzes the starch and thepH buffer, the experiment still shows that amylase works best at slightly acidic, or neutral,conditions. Basic conditions and both extreme pH levels are shown to denature the enzyme.Some aspects of the experiment may have yielded some inaccurate data. For example, thetime interval at which the mixture of starch and enzyme solution were added to the well maynot have been very accurate. Human error may have occurred at watching the stopwatch andadding the mixture. In addition, the enzymes probably do not always exactly hydrolyze at 30-second intervals, therefore the large time difference between each interval may havegeneralized the data too much. This, thus, may be responsible for the identical duplicateresults. In other words, if the time intervals were less wide, the reaction time may have beenmore specific, and therefore not as identical for each trial.In addition, only five pH buffers were used for this experiment. This may have generalizedthe results as well. For example, slightly basic conditions could have allowed starchhydrolysis, or slightly acidic conditions may have allowed even more optimal enzymatic
activity than pH buffer 4.Finally, the imprecise amount of stirring that applied to each well and each time interval mayhave yielded unreliable data. For example, because the solutions were very sensitive duringreactions, a harder stir at one well may have speeded up the reaction.Tabulated below are the aspects of the experiment that could have affected the accuracy ofthe results. Error Impact Improvements Shorten the time between each interval Generalized the data For example, take 10 Inaccurate proof of the seconds in between time taken for the starch to each interval in order 30-second interval be hydrolyzed to find out more Inaccuracy in adding the accurate results about solution at exactly each the time it takes for the time interval starch to be completely hydrolyzed Generalized the data about which pH level affects Use more pH buffers enzyme activity Use more than 2 acidic Insufficient number of pH Less accuracy in pH buffers buffers determining the exact Use more than 2 basic conditions that support pH buffers enzymatic activity Set a constant number for May have yielded biased inserting and extracting of data solution with the Equal stirring for each well Uneven stirring for micropipette different wells may have This would add more yielded different data uniformity at each well of the microplate