Enzymatic Activity of Salivary Amylase

Punsalan, Gian Domini M. 3Bio-6, Department of Biological Sciences, College of Science, University of Santo Tomas, Espana, Manila

Abstract Salivary Amylase catalyzes, digests and hydrolyzes Starch Molecules into simpler compounds factors that can affect the rate and specificity of Salivary amylase are temperature and PH. Rates of Salivary Amylase enzymatic activity and varying

temperatures and PH were measured. The measurements were compared by plotting the points in a graph.

I.

Introduction

Enzymes are biological catalysts or assistants that speed up the reactions occurring in living organisms. Enzymes drive the chemical reaction faster for a specific reaction or nutrient. The chemicals that are transformed into useful substrates are called substrates. The site where substrates are transformed is called an Active Site. Substrates are held by non-covalent interactions. Enzymes can speed up the reactions at the rate of ten times per second. Enzymes work by lowering the activation energy, thus speeding up the reactions. Enzymes are known to catalyse about 4000 biochemical reactions. Enzymes are crucial for survival.

Salivary amylase begins the process of digestion. They break down starch and sugar. Food contains carbohydrates and proteins that need to be broken down and converted into energy. Salivary Amylase breaks down starch into a disaccharide, Maltose. The objectives of this experiment is to examine the enzymatic activity and specificity of salivary amylase depending on changes in pH and temperature and to determine the narrow range of pH values and different temperatures at which enzyme exhibits its optimum activity.

II.

Materials and Methodology

The materials and reagents used in the activity which examines the rate of enzymatic activity using varying temperatures are an Enzyme solution (1mL saliva + 9mL distilled H2O + 30 mL 0.5 % NaCl), Buffered starch (1% starch in phosphate buffer pH 6.7), 0.001 Iodine Solution, 2 Spot plates, test tubes, medicine droppers, and constant temperature bath (4, room temp, 37,50,60 and 70C). The materials and reagent used in the activity examines the rate of enzymatic activity using varying pH are an Enzyme solution (1mL saliva + 9mL distilled H2O + 30 mL 0.5 % NaCl), 2% Unbuffered starch, 0.001 M Iodine Solution, 2 spot plates, large test tubes, medicine droppers, water bath set at 37 c, Acetate Buffer solutions (pH 4 and 5), Phosphate buffer solutions (pH 6.7 and 8), Bicarbonate Buffer (pH 10).

For the effect of temperature, 2 mL of the enzyme solution was placed in a large test tube. This was labelled 4C. 2 mL of the buffered starch solution was added in a separate large test tube. Both test tubes were incubated for 10 minutes in an ice bath (4C) Three drops of mixture was immediately mixed and quickly taken and two drops of the iodine solution were added onto a spot plate simultaneously. This was the zero-minute.

After a one minute interval, three drops of the mixture was taken and two drops of the iodine solution was added onto the second well simultaneously. This was the one minute. This was repeated until light yellow-colored reaction was observed. The time was noted. For the other temperatures, all steps were repeated following the desired incubation temperature. The

reciprocal of time versus the temperature was plotted. The optimum temperature of the amylase was determined.

For the effect of pH, 1 mL of acetate buffer (pH 4) and 1 mL 2% unbuffered starch was placed in a large test tube. In a separate test tube, 2 mL of the enzyme solution was added. Both test tubes were incubated for 10 minutes in a 37C water bath. Three drops of the mixture was immediately mixed and quickly taken and two drops of the iodine solution was simultaneously added into the first well. This was your zero minute.

After one minute interval, three drops of the mixture was taken and two drops of the iodine solution was added simultaneously onto the second well. This was your one minute. The last step was repeated until a light yellow colored solution was observed. The time was noted. For the other pH, steps 1 to 6 were repeated using an appropriated buffer. The reciprocal of time versus the pH was plotted. The optimum pH of the amylase was determined.

III.

Results and Discussion

A. Effect of Temperature

Temperature (c) 4 Room Temperature 50 60 70 80

Time (min) 6 16 30

1/t (min-1) 0 0.167 min-1 0.0625 min-1 0.033 min-1 0 0

Temperature
0.18 0.16 0.14 0.12 0.1 0.08 0.06 0.04 0.02 0 0 10 20 30 40 50 60 70 80 Temperature

Table 1. Temperature vs. Reciprocal time

The graph above shows the reaction rate of the enzyme salivary amylase using varying temperatures. The results show that in increasing the temperature, the reaction rate would speed up but only up to a maximum. This is called the optimum temperature. The tests show that 37 degrees Celsius is the optimum temperature which means that this is the maximum temperature that would speed up the reaction. Increasing the temperature above the optimum temperature would make the reaction speed up but some enzymes are denatured. Heating the enzymes gives the protein extra energy to move more. As the temperature rises, molecular motion and large collision between substrates speed up but enzymes are proteins; thus, having a limit beyond which the enzymes become denatured. Given enough time and higher temperature, the enzyme structure would be broken permanently. Different enzymes have different optimum temperatures. Salivary Amylase based on the results has an optimum temperature of 37 degrees Celsius.

A. Effect of pH

pH 4 5 6.7 8 10

Time(min) 16 1 16 18

1/t (min-1) 0 0.0625 1 0.0625 0.056

pH
1.2 1 0.8 0.6 pH 0.4 0.2 0 0 -0.2 Table 2. pH vs. Reciprocal of Time 2 4 6 8 10 12

The graph above shows the reaction rate of Salivary Amylase in varying pH values. It exhibited a bell-like structure. The results show that there is also a peak in pH values where enzymes work best. This is called the optimum pH. The table and the graph above shows that ph 6.7 works best in speeding up the reactions of Salivary Amylase. Using lower or higher pH values could lead to the breaking of the bonds that holds the structure of the enzyme in place. The enzyme could lose its shape and the shape of its active site that would not be fit to the substrate. Going above the optimum pH of the salivary amylase which is 6.7-7 can break the tertiary structure of the enzyme. The enzyme, as a protein, is crucially dependent in its conformation.

Starch is catalysed by to enzyme solution in this activity. The Iodine solution was used to detect the presence of starch. This test was applied to observe the rate of loss of reactant or increase in product per minute. Sodium Chloride was added in the enzyme solution to activate the salivary amylase.

IV.

Conclusion

The experiment emphasizes the factors that can speed up the reaction rate of enzymes, particularly Salivary Amylase. There is an optimum temperature and optimum pH in which the enzyme works best. Not using the optimum temperature and optimum pH could destroy the structure of the enzyme and could cause enzyme denaturization. The optimum temperature of Salivary Amylase is 37 degrees Celsius and its optimum pH is 6.7.

V.

References

Menguito, C.A. (2010). Basic Laboratory Experiments in Biochemistry. UST College of Science. Pratt, C. & Cornely, K. (2010). Essential Biochemistry. John Wiley and Sons, Inc. http://www.chemguide.co.uk/organicprops/aminoacids/enzymes2.html http://www.worthington-biochem.com/introbiochem/tempeffects.html http://www.worthington-biochem.com/introbiochem/effectsph.html