This experimentation was to evaluate absorbance and the reaction rate of an enzyme, '-amylase in starch-iodine solution. We will be testing the relationship between enzymatic reaction affected by temperature and pH. Through the testing the enzyme at different temperatures, and different pH levels; it would determine at which temperature and pH level the enzyme worked the most efficiently. Analyzing absorbance of the solutions with spectrophotometery will determine the reaction rate.
To test the optimal pH, the starch and a buffer were combined at a specific pH level and tested the absorbance of a solution at various times. To resolve the reaction rate of a solution at various temperatures, the solution was put into water both set to a specific temperature and its absorbance was recorded at different times.
Four specific graphs were drawn to determine at which pH level and temperature was the optimum for the enzyme after the experiment.
Although hypothesis that I made that it would work best under temperature near 40'' and at a pH of around 4, through my results the enzyme worked more efficiently under the temperature of 55''and pH level at 4.5. However, since the whole testing experimental was taken only once; we can not state that the result was precisely accurate. Introduction: The catalysts are the substance that speeds up chemical reactions in living organisms. The enzymes are the protein catalysts to lower the activation energy of a reactant. Enzymes are not able cause an inefficient reaction to occur and only able to increase the rate of biochemical reaction. The molecule that is reacting in enzymatic reaction is called a substrate. When an enzyme and substrate congregate together, then it forms an enzyme-substrate complex. As the enzyme works to accelerate the chemical reaction changes catalytic property and ultimately it decomposed.
The rate of the enzymatic reaction is when the rate at which the enzyme-substrate complex forms and it decomposes to form the product. There are two factors that formulate enzymatic reaction. The first factor is that time required by the enzyme to alter the substrate to its product, and the second factor is the rate that it takes to form the enzyme-substrate complex. A typical enzymatic reaction shows a fading of the substrate after an extended period of time. The substrate becomes a part of the product during this reaction, diminishing the probability that an enzyme molecule would have during the reaction, reducing the chances that an enzyme molecule would have to meet with substrate molecule leading to a decrease of the reaction rate. Temperature and pH would be the other factors that related with a change in the rate of an enzymatic reaction. At particular pH and temperature levels, the enzyme performs its best but if the two factors are either excessively high or excessively low, it can cause the enzyme to denature and diminishing the reaction rate.
In this research, we used the enzyme '-amylase, where found in both plants and animals. Amylase hydrolyzes starch, glycogen, and dextrin to form glucose and maltose. (Brewing 2002) In animals '-amylase is digestive enzymes that made from mainly within the pancreas and salivary glands to break down starches form the nutrients. (Great Vista Chemicals, 2005) Human amylase has an optimum pH of 7.2-7.4, and denatures at temperatures above 60??. (Research Machines, 2005) With the warm condition enzymes like '-amylase have been said to work the most efficiently, which is around 40??. It was also stated that they work best in the acidic rage from 3.5 to 5.5. (Thermophilic Moulds in Biotechnology, 1999)
With preceding experiments, a hypothesis can be stated that the optimal pH of '-amylase would occur m at a temperature near 40'' and pH at around 4. This experiment would examine the hypothesis and would confirm the optimal of both the temperature and pH of the enzyme '-amylase. Materials and Methods:
This laboratory research is composed of two main experiments, the first research to examine the starch-iodine solution at different pH levels: 4.0, 4.5, 5.0, 6.0, 6.5, and the second research to determine the starch-iodine solution at different temperatures: 15'', 30'', 45'', 55'', 60'', 70??.
Before the proceeding of an experiment, makes sure the spectrometer turned on for at least 15 minutes to be warm. After turning on the spectrometer set the wavelength at 560nm. Add '-amylase to starch-iodine solution. To examine the starch-iodine solution at different pH levels, a 35mL stock starch and 35mL of buffer at pH level 4.5 were placed into an Erlenmeyer flask to set up the reaction flask solution. Adjust spectrophotometer with mixture of 5mL of distilled water and 0.1mL of the starch iodine in cuvette (the blank). Turn the transmittance knob and set the line at zero. Place the ready blank solution into the spectrophotometer and turned the absorbance knob and set the line at zero. Then added 0.1mL of the I?'KI indicator into the cuvettes.
Record the first data at 0 minute, add 5mL of the solution from the reaction flask into a cuvette with I?'KI indicator in it. Then placed cuvette into the spectrophotometer and recorded the absorbance. After recording the initial absorbance at 0 minute the TA put 1mL of '-amylase solution into the reaction flask then began to time the experiment. Every two minutes 5mL of solution was put into starch-iodine solution. The absorbance was recorded every two minutes for 20 minutes. After recording the absorbance of 35mL of distilled water and the 35mL buffer at a pH of 4.5, remaining pH levels followed same procedure.
The experiment of the solution at different temperatures requires almost same procedure but one more step is added. Prepare another solution consisting of 35mL of distilled water and 35mL starch-iodine solution and placed them into an Erlenmeyer flask. The Erlenmeyer flask was then placed into a water bath with the temperature set to 45??. Make sure the water bath is warm. The spectrophotometer was adjusted with the blank which contained 5mL of distilled water and 0.1mL of I?'KI. Then set the wavelength of spectrophotometer at 560nm. Adjust spectrophotometer with mixture of 5mL of distilled water and 0.1mL of the starch-iodine in cuvette (the blank). Turn the transmittance knob and set the line at zero. Place the ready blank solution into the spectrophotometer and turned the absorbance knob and set the line at zero. Then added 0.1mL of the I?'KI indicator into the cuvettes. Record the first data at 0 minute, add 5mL of the solution from the reaction flask into a cuvette with I?'KI indicator in it. Then placed cuvette into the spectrophotometer and recorded the absorbance. After recording the initial absorbance at 0 minute the TA put 1mL of '-amylase solution into the reaction flask then began to time the experiment. This time every one minute 5mL of solution was put into starch-iodine solution till 8 minutes put 5mL of solution at 10 minutes, then put another 5mL of solution at 20 minutes. The absorbance was recorded every one minutes for 8 minutes and at 10 minutes and at 20 minutes. After recording the absorbance of 35mL of distilled water and the 35mL buffer at a temperature of 45'', remaining temperature levels followed same procedure.
Two graphs were made when the all the data were written down into two different charts. The first graph was absorbance of reactions at different pH, and second graph was absorbance of reactions at different temperatures. On the graph y-axis indicates the absorbance that is measure by the time which indicated on x-axis during the experiment. The graph showed how the absorbance changed within the period of time.
Then the reaction chart had to be completed. First filling out initial and final absorbance for the both different temperature levels and different pH levels, find out the delta of absorbance(?'A) and 1/2 of delta absorbance. Then subtract 1/2 of delta absorbance from initial absorbance, and write down the time that 1/2 delta absorbance took. After writing down the time the reaction rate was calculated using this equation: (??'A)/(TAi-??'A).
When the reaction rate chart was completed two more graphs were drawn using those values. The reaction rate at different temperatures and different pH levels, the x-axis indicated the temperatures and the pH levels, the y-axis indicated the reaction rate in absorbance per minute.
The first graph figure 1 shows that all of the absorbencies were decreased over the time. The highest starting absorbance was pH level at 4.5, and pH level of 4.0 started with the lowest absorbance. At pH level 5.0 and 5.5 the graphs shows some common. The pH level at 4.5 shows great change in slopes, and the pH level at 4.0 shows least change.
The second graph figure 2, similar graph has been drawn with the first graph. The absorbance of the different temperatures gradually reduced over time. At 15'' it started with the highest absorbance and at the 70'' it started with lowest absorbance. In the figures 2, it seems that all the absorbencies started off from lower absorbance than it was in figure 1. It also shows that at 60'' and 45'' graphs are very similar.
The third and the last graphs show the reaction rates. In the figure 3 and 4 the graphs have a shape of bell with peak point. This is the optimal point of the reaction rate. The graph starts with low reaction rate and it increases then it hits the peak point and it decreases to lower reaction rate. The optimal point for the reaction rate at different temperature was at 55??(Figure 3) and the optimal point for the reaction rate at different pH level was at 4.5(Figure 4). Discussion:
Before this laboratory experiment, I had a base knowledge that after putting the enzyme, '-amylase into a starch-iodine solution; I knew the substance concentration would decrease as the time passed. Also I made a hypothesis that under the condition of temperature at 40'' and pH at around 4, the enzyme works at best efficiency. The result was little bit off but quite similar to what I expected from my hypothesis. The first two graphs (Figure1, Figure2) shows obvious decrease in absorbance as the times increases. The rest of two other graphs (Figure3, Figure4) show how the reaction rates are different at various temperatures and pH levels. Within the information given, my hypothesis of optimal temperature of '-amylase would occur at 40??. The actual graph shows (Figure3) that optimal rate was at 55??. By looking at the graph in Figure 2, I could tell that 55'' has the stiffest slope out of all different temperatures. This result was 15'' off from what I expected from my hypothesis. For the optimal pH level, the graph in Figure 4 shows that optimal point is at 4.5. This was very close with my prediction since my hypothesis stated the optimal pH level at around 4.
Since, we've done this laboratory experimental only once I could not declare that the result was accurate. Also, an error might have been occurred during this experiment. Those errors occurred from lack of laboratory experiences. One example of lack of laboratory experience could be inaccuracy from the timing. Since the reaction changes every second, if the solution was add too early or too late it would made difference in the result. Another error would occur from inaccurate volumetric readings. Since, some of the measurement was read by human eye it can not be accurate, also the might have miss read the measurement.
My over view from this laboratory experiment is very successful. The hypothesis and the actual result were close enough to state that my hypothesis was right. The '-amylase enzyme works best at warm temperature around 40'' and acidic pH level around 4.5.
Literature Cited: Amylase Enzyme. Great Vista Chemicals URL:(www.greatvistachemicals.com/biochemicals/amylase.html) B N Johri, T Satyanarayana, J Olsen. 1999. Thermophilic Moulds in Biotechnology. Kluwer Academic Publishers. AA Dordrecht. Netherlands. Michael Lewis, Tom W Young 2002. Brewing. Kluwer Academic/Plenum Publishers New York, New York. Research Machines. Plc 2.5 Helicon Publishing. URL:(www.tiscali.co.uk/reference/encyclopedia/hutchinson/m0007936.html) Vliet, K.A.(ed.). 1996. A Laboratory Manual for Integrated Principles of Biology: Part One BSC2010L. Ginn Press, Needham Heights, Massachusetts.