Assessing reaction speed of the enzyme amylase can be measured by the amount of glucose and maltose produced during given time intervals. I hypothesized that, if the reaction time is longer, then the amount of amylase will be larger. Enzymes are specific in their match of substrates they will breakdown – similar to a key and its lock. Since amylase is the only enzyme that breaks down starch, the procedure was effective and gave clear results of sugar produced. With help from the stain reagent benzoic acid – I visibly saw the production of sugar molecules.
However, I was only able to determine which of the Tubes experimented had the most or least sugar present, by color intensity. By operating a spectrophotometer, I was able to create a standard curve, which gave the absorbance values of glucose/maltose concentrations in the Tubes tested. From that, using the standard curve results and calculations (7. 8 x 10^16) I then determined the µmoles of sugar present in each Tube.
Finding that, in at least 2 minutes, enzyme amylase will have broken down enough starch to obtain 0. 26 µmoles of sugar.
Introduction Enzymes are proteins that act as catalysts. Enzymes break down big molecules like starch into small molecules like glucose, so that they diffuse through cell walls in our body into the blood stream. It has to be the correct enzyme – amylase is the only enzyme that will break down starch. Starch is a polymer composed of a series of glucose monomers. Amylase hydrolyzes starch into glucose and disaccharide maltose (Lab pg62). The lock and key theory is a possible explanation of why, only one type of enzyme can breakdown starch.
The analogy is that, the enzyme is the lock and the key is the substrate, only the correct key (substrate) fits into the keyhole (active site) of the lock (enzyme). Utilizing a stain reagent (benzoic acid) that does not react with starch and deactivates the enzyme amylase, will indicate after heated – the most to least sugar present. To clarify the amount of sugar in each tube, can be found using a spectrophotometer. A spectrophotometer measures light absorbance and requires cuvette tubes, which are smaller than regular tubes, and have a white line indicator below the lip.
Cuvettes need to be wiped clean for accurate results and they must contain stained sugar molecules to obtain an absorbance above zero. Once the absorbance values are known, a standard curve can be made to represent the connection between the amount of sugar in a tube and the corresponding absorbance. To establish the µmoles of sugar present in each tube can be found using the standard curve. Materials and Methods Procedure: wear safety glasses and gloves – stain is highly reactive – wash off skin immediately if spilled on, and inform instructor.
Part A: Using an enzyme deactivating stain reagent that doesn’t react with starch, I was able to determine which sample of the 6 had the most to least sugar present. Beginning with 5 test tubes – labeled 2, 4, 6, and 6C – to each one I added 10 drops of 1% starch (pH7. ) The last tube I labeled “B” to represent a blank (control), in which I added 0. 5mL of the stain (color reagent,) and 2 drops of the enzyme solution. Starting the amylose reaction I added 2 drops of enzyme solution to Tube 6 noting the exact time. Precisely two minutes later, I added 2 drops of enzymes to Tube 4.
Two minutes later, I added 2 drops of enzyme to Tube 2. Meaning Tube 6C is without enzyme solution at this time to indicate the amount of starch broken down during the six minutes, if any, without the enzyme present. Six minutes after adding enzymes to Tube 6, using a 1 mL pipet, I added 0. 5 mL of stain to each of tubes 6, 6C, 4, and 2 – stopping enzyme activity – with the exception of Tube 6C. Then in Tube 6C, I added 2 drops of enzymes. After mixing the contents of each tube, using a hot plate and beaker of water – I boiled the tubes for five minutes.
I removed the tubes to then add 2. 5 mL of dH2O to each of them using a 5 mL pipet. Mixing contents in each tube till the color was uniform to show the amount of starch breakdown after six minutes. Part B: By observing the color intensity of the sugar molecules from darkest to lightest – I was able to rank the tubes in terms of most to least sugar present. Next, to determine the light absorbance for each tube, I transferred the contents of each tube to a corresponding numbered set of the smaller tubes (cuvettes. Then, to measure the light absorbance of sugar molecules present, I utilized a spectrophotometer. Following was adjusting the spectrophotometer to a wavelength of 540 nm, and setting the meter to 0% transmittance. Prior to placing the cuvettes in the chamber, I had to be sure to wipe off any fingerprints or residue that may impede the proper function. With the spectrophotometer set on absorbance mode, I inserted each of the 7 cuvette tubes in the closed chamber.
Part C: After recording each tubes absorbance made by the present stained sugar molecules, I created a graph of the standard curve. In the graph I included the relationship between the quantity of sugar and the degree of absorbance. Results After completing Part A, I was able to determine which tube of the 5, had the most and least sugar present. Since the stain (color reagent) deactivates amylase, test Tube B did not breakdown starch or produce sugar. Therefore, the dark color is only due to the stain and hydrolysis of starch did not occur at all in test Tube B.
Test Tube 6C represented a reaction in which enzymes were added after the stain regiment – which stops enzyme activity – to see if starch would in fact breakdown without enzymes present. Tube 6C was dark in color, but not as dark as Tube B, so some hydrolysis did in fact occur. Tubes 2, 4, 6 represented the reaction times of 2, 4, and 6 minutes correspondingly. I found that Tube 6 was the darkest of the three, and also processed the longest. Tube 4 was somewhat lighter than Tube 6, and Tube 2 of course, was lighter than the both.
Since, the amount of sugar in the Tubes was still unknown at this point. Subsequently, the use of the spectrophotometer in Part B – allowed for a clearer view of the actual amount of sugar molecules present. As seen in Graph 1 (standard curve), the amount of µmoles of sugar increased as did the absorbance. Once the absorbance was known, I was able to calculate (7. 8 x 10^16) the amount of sugar it contains from the standard curve as seen in Graph 2. Graph 1: Graph 2: Discussion All in all, the longer the reaction time, leads to increased amounts of amylase activity.
In other words, the longer the enzyme amylase is able to work at hydrolyzing starch, the more sugar will be produced. This was concluded only after absorbance values of each Tube were calculated from the spectrophotometer. The spectrophotometer provided an exact number of stained sugar molecules as opposed to determining sugar content by darkest and lightest color in Part A. I can conclude that, in order for sugar to develop by the breakdown of starch, it needs to sit for at least 2 minutes without the addition of the stain reagent benzoic acid.
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