The Effect of Temperature on Enzymatic Activity of Amylase - Lab Report Example

A Study to Examine the Effect of Temperature on Enzymatic Activity of Amylase” Enzymes are biological catalysts, that are present in the body to carryout various metabolic activities. They are basically made up of protein molecules which are very sensitive to temperature fluctuations in the surrounding where they accomplish biological reactions. Our daily diet encompasses great quantity of starch. Hydrolysis of starch is executed by amylase, an enzyme that acts on starch to break it down into maltose, maltriose, short oligosaccharides. This paper determines the enzymatic activity of amylase over a range of temperature and thus brings a close look on the optimal temperature for amylase activities in human (human amylases) and fungus, Aspergillus oryzae (fungal amylase). The temperature range was set for 0°C to 95°C. the rate of reaction was directly proportional to the increase in temperature, but there was a decline after 40°C as envisaged in the hypothesis, the enzymes denature with a very high elevation in temperature, concluding that if the temperature is increased beyond the optimal temperature then the enzymatic activities come to a close and enzyme becomes denatured. The study also envisaged that fungal amylase do display the slightly faster hydrolytic action on starch as compared to the human salivary amylase. Introduction Starch is the regular component of our daily diet. It is perhaps an extensive carbohydrate in the plant world and is consumed by humans and other herbivores to meet their energy requirements. It is a polymer of glucose which depicts the linkage of α-1,4 and α-1,6 glucosidic bonding. It is essential to break this bonding to derive carbon and energy from the starch molecules; this task is performed by group of enzymes called amylases (Lehninger, 2008). There are various organisms which serve as sources of amylase, human saliva and pancreas, other organisms encompass plants fungi and bacteria. The present study includes two group of amylases, human amylase and fungal amylase. Human amylase was obtained from the saliva while the fungal amylase was procured from Aspergillus species which is highly prevalent and generally acquired from soil, putrefying foliage and also present in air. Any reaction needs some initiation energy for its onset; this is also true for biological processes. Hydrolysis of starch also requires some activation energy but due to the presence of biological catalysts, enzyme amylase this activation energy can be reduced and reaction is carried out with ease. Enzymes are extremely specific for substrates, pH, temperature. This is explained with the fact that every lock opens with its own key hence “Lock & Key” model explains the mechanism of action for enzymes. Substrate binds to the active site of the enzyme, making an enzyme-substrate complex, which is essential to for breakage and reunion of bonds leading to the formation of product(s) releasing the enzyme in its initial shape (Lehninger, 2008). It is known that proteins denature at higher temperature, enzymes are protein molecules and therefore they are liable to be denatured at higher temperature rendering the enzyme inactive due to trouncing of its three-dimensional structure (Lehninger, 2008). The present work is carried out on this hypothesis; the amylase activity essentially increases with the raise in temperature and more of the substrate is being converted to the product but as the temperature becomes excessively high enzymes are denatured and substrate can no longer bind to enzyme. The study scrutinizes the effect of variation in temperature on the action of amylase over the starch molecules and takes account of finding the optimum temperature for amylase activity in case of human amylase and also fungal amylase. Materials & Methods Amylase solution, 1% Starch solution, Iodine solution, Spot plates, test tubes, test tube stand, Thermometer, Glass pipettes, Stop watch, Water baths at 40°C, 60°C, 95°C. In case of H-amylase, saliva must be collected. Before saliva collection, rinse mouth well with water, in case if it is difficult to collect enough saliva then chew parafilm to produce good quantity of saliva. Experimental Setup: 1. Take 2 spot plates with 12 wells/ spots each (4x3). Join the plates so as to make 10 rows (mark them for time duration 2, 4, 6, 8, 10 mins) and 4 columns for the temperature range (0°C, 40°C, 60°C, 95°C). 2. Take 4 test tubes for 4 different temperatures and take the enzyme source. Mark the test tubes for temperature and H-if amylase source is human or F- if amylase source is fungal. Mark the group number on the test tubes. 3. Take another set of 4 test tubes for 4 different temperatures, with enzyme source (H/F), mark the group no. and S for starch solution. 4. 5ml of 1% starch solution was placed in 4 test tubes. 5. For H-amylase, 0.5ml of saliva was placed in each test tube which was diluted with 0.5ml of distilled water. 6. For F-amylase, 1ml of fungal amylase solution is added to each tube. 7. Marked test tubes were placed in their respective temperature zones. For 0°C into ice bath, for 40°C in the water bath set at 40°C, for 60°C in water bath set at 60°C and for 95°C in the water bath set at 95°C. 8. The contents of the two sets should not be mixed but must be allowed to equilibrate in their respective environments/ temperature zones for 5 mins. 9. After leaving the tubes, put 2 drops of iodine solution in the set spot plate. White paper must be placed below the spot plates to get the good visibility of the reaction. 10. As soon as the equilibration process is ended, transfer a few drops of starch solution from each temperature treatment to the first row of the spot plate that is marked as 0 min without removing the tubes from water bath. Separate pipette must be used for every temperature handling. For convenience mark the pipette with respective temperature. 11. Dispense the starch solution into the tube containing amylase. The timer must be set for 2 min during the amylase addition. 12. Using the respective pipettes, place 2-3 drops of the starch-amylase mixture on the second row of the spot plate marked as 2 mins under their respective temperatures. Depending upon the amylase source color change is monitored and recorded in observation tables. If starch is present the color is deep blue if it is hydrolyzed then it gives pale yellow to brown. 13. The process must be repeated for 10 mins and 100% hydrolysis must be recorded against the temperature zone. 14. The color code scheme must be used to convert the qualitative data into quantitative data. Observation Table: Temperature Time (min) 0°C H 0°C F 40°C H 40°C F 60°C H 60°C F 95°C H 95°C F 0 min 2 min 4 min 8 min 10 min Results The results procured do support the hypothesis, which state that with the increase in temperature the temperature increases, but at a very elevated temperature the enzymatic activity cease and there is no color change indicating that the starch was not hydrolyzed which directly imply that the enzyme, amylase got denatured and lost its effect. The results follow the pattern as hypothesis says, at 40°C the rate of reaction was maximum as there was a rapid change in color and no starch was found the color was pale yellow which indicates that the starch is hydrolyzed. This steep rise in the rate of reaction was followed by a steep fall at 60°C and at 95°C the enzyme was totally denatured and so no activity was recorded. The graph is giving the similar look as was expected and also correspond to the graphs depicting effect of temperature on the rate of reaction (Lehninger, 2008). The result signify that amylase can tolerate temperature of 60°C with maximum activity at 40°C which is recorded as the optimum temperature in both the cases H and F. Discussion The results indicate that the hypothesis developed was true in the sense that reaction would increase with the increase in temperature until the temperature is reached where the activity of the enzyme ceases. This gives a bell shaped curve depicting that enzyme activity do show a lag phase and a log phase with the increase in temperature, this log phase is due to enhanced kinetic energy of substrate and enzyme. The kinetic energy corresponds to the motion of the molecules and hence both, enzyme and substrate molecules moves faster and collide at a rapid rate to give towering to the rate of reaction. This depicts that temperature do affect the amylase activity and enhances the enzymatic action up to an optimal temperature level and gradually when the temperature is further increased then the secondary and tertiary protein molecular structures are lost eventually leading to the denaturation of protein at 95°C. When the results of H-amylase and F-amylase are compared the rate of reaction was faster in case of F-amylase, which was monitored with the rapid change in the color of iodine solution. This indicates that the optimal temperature for both these enzymes although do not show much variation but the rate of action to hydrolyze starch into maltose is executed by fungal amylase at a faster pace than the human salivary amylase, concluding that fungal amylase can break starch easily. In case of H-amylase the activity of enzyme is best at 37°C which is the normal body temperature. The salivary amylase works at neutral pH and therefore to avoid any kind of errors in the results precautions regarding thorough cleaning of the glassware is essential. References: 1. Lehninger Principles of Biochemistry, 5th Edition, Chapter 6. W. H. Freeman. 2008. Read More