MethodologyA. Preparation of Starch Standard CurveDifferent concentrations of starch solution in different 10 ml test tubes were prepared by following the table below. The 2ml of each solution were taken and were diluted to 10.0ml. The different concentrations of starch solution were given I2 solution just before they were measured for absorbance. The absorbance of each I2-starch complex were measured at 620 nm. The data was then plotted as starch concentration vs. absorbance.

Table 1: Different concentrations of starch solution (Manila, 2013)Test tube #Volume of 0.1% starch solutionVolume dH2O (ml)Volume I2 solution (L)Starch Concentration (%w/v)

10.003.0020.0

20.502.5020.0

31.002.0020.0

41.501.5020.0

52.001.0020.0

62.500.5020.0

73.000.0020.0

B. Reaction of Amylase in Human SalivaThe amylase of 100 ml of 1:10 dilution ratio of human saliva was prepared in a conical tube. In five separate test tubes, the 1 ml of enzyme solution and the 9 ml of 0.1% starch solution were mixed. The different test tubes were incubated at room temperatures for 0, 3, 5, 7 and 10 minutes respectively and all were immersed in boiling water for 3 minutes. The tubes were cooled and were given 2 ml distilled H2O and 1 drop of I2 solution. The 2 ml of each solution were taken and diluted to 5 ml. The absorbance of the solutions were measured at 620 nm. The starch concentration vs. the incubation time was graphed by converting the absorbance measured into starch concentration using the standard curve. The average reaction velocity was calculated by getting the change in starch concentration over the change in time per interval. The reciprocal of the starch concentration vs. the reciprocal of the average reaction velocity was graphed to determine Km and Vmax.C. Effect of temperature on Enzyme activityThe following were mixed in each of the four separated test tubes: 1 ml of 0.1% starch solution, 3.5 ml of 0.1 M phosphate buffer of pH 6.7, and 0.50 ml of 0.9 % NaCl solution. Each of the test tubes were pre-incubated separately in different water baths with temperatures of 10 C , 40 C, 60 C and 80 C for 5 minutes and all were added 5 drops of enzyme solution and all were continued incubation for 10 minutes more. The four test tubes were immersed in a boiling water for 3 minutes and were cooled to room temperature. An ml of an aliquot of each test tubes were taken and were given 2.0 ml of distilled H2O and a drop of I2 solution before measuring for absorbance at 620. The absorbance data was converted to starch concentration was graphed vs. temperature.

D. Effect of pH on Enzyme ActivityIn each four separated 10ml test tubes, the following were mixed: 1ml of 0.1% starch solution and 0.50 ml of 0.9% NaCl solution. Different pH (4.0, 6.7, 7.4, 9.0) of 3.5 ml 0.1 M phosphate buffers were added respectively on each of the four separated test tubes. All tubes were added with 5 drops of enzyme solution and were incubated at room temperature for 10 minutes before immersing the tubes at boiling water for 3 minutes. The test tubes were cooled and 1 ml aliquot of each test tubes were mixed with 2 ml of distilled H2O and a drop of I2 solution. The absorbance of each mixture were measured at 620 nm and converted into starch concentration. The data gathered was graph into starch concentration vs. pH.

IntroductionEnzymes are the catalyses of the reactions undergoing in our body. They speed up reactions by finding an alternative reaction pathway, lowering the activation energy and stabilizing the intermediates in the reaction. (Science, 2004).

Figure 1: general equation of enzymes (Massachusetts, 2015)

There are two hypotheses behind how enzymes work. These are the lock and key hypothesis which states that the substrate simply fits into the active site to form a reaction intermediate and the induced fit hypothesis which states that the enzyme molecule changes shape as the substrate molecules gets close. The change in shape is 'induced' by the approaching substrate molecule. This more sophisticated model relies on the fact that molecules are flexible because single covalent bonds are free to rotate. (Massachusetts, 2015)

There are four factors which affects the catalytic activities of enzymes. They are temperature, concentration of the enzyme and substrate, pH and the presence of inhibitors. The purpose of the experiment is to find out how these factors affect the activities of the enzyme amylase which are found in the human saliva and compute for the affinity constant of the sample saliva and its maximum reaction velocity at different situations by using the Michaelis-Menten equation and the Lineweaver-Burke equation. (J.H, 1971)

Figure 2: Michaelis-Menten Equation (Michaelis-Menten Kinetics and Briggs-Haldane Kinetics, 1925)

Figure 3: Lineweaver-burke equation (Enzyme Kinetics as an Approach to Understanding Mechanism, 2011)ReferencesEnzyme Kinetics as an Approach to Understanding Mechanism. (2011). Retrieved from Bioinfo.org: http://www.bioinfo.org.cn/book/biochemistry/chapt08/sim2.htmJ.H, W. (1971). Enzyme kinetics and its relevance to enzyme assay. Journal of Clinical Pathology, 14-21. Retrieved from http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1176280/?page=2Manila, U. o. (2013). Laboratory Manual in Biochemistry. Manila.Massachusetts, U. o. (2015). Intro Biology. Retrieved from UMassAmherst: http://bcrc.bio.umass.edu/intro/content/enzyme-kinetics-lab-protocolMichaelis-Menten Kinetics and Briggs-Haldane Kinetics. (1925). Retrieved from Michaelis-Menten Kinetics and Briggs-Haldane Kinetics: http://depts.washington.edu/wmatkins/kinetics/michaelis-menten.htmlScience, R. A. (2004). Enzymes. Retrieved from RSC: http://www.rsc.org/Education/Teachers/Resources/cfb/enzymes.htm