Enzymatic Activity of Salivary Amylase

Bagual, Christlee Jill V. Chemistry Department, College of Science, Adamson University, Ermita, Manila

ABSTRACT
Enzymes are proteins that act as catalysts for different metabolic reactions for an instance in catalyzing starch to disaccharide by the activity of salivary amylase. Enzymatic activity consists of substrate, catalyst and product. Catalysts have active site where catalysis occurs but enzymes activity was greatly affected by different factors such as temperature and pH. Specificity of salivary amylase was measured and the result gave an optimum temperature at 37C and pH at 7 wherein at this point the reaction of iodine with starch gave a yellow coloration. Keywords: enzyme, catalyst, salivary amylase, active site

Introduction
Living systems were designed by huge variety of biochemical reactions which are all mediated by enzymes. Enzymes are made up of largest and most diverse group of proteins which acts as biological catalyst. They cause to accelerate the rate of chemical reaction by lowering the activation energy or free energy barrier (see Figure 1) that separates the reactants and products (Voet et al., 2008). Smaller molecules known as substrates were the specific target where enzymes act to produce a product. The relationship between substrates, enzymes and products can be represented by the equation (CH102Labs, 2005): ENZYME SUBSTRATE ---------> PRODUCT Mechanisms of enzymes depend on the conformational arrangement of its enzymes active site, the region where catalysis occurs (Voet et al., 2008). Each metabolic reaction has to be catalyzed in the living organism by its own special enzyme. During digestion enzymes were released into the mouth, stomach and intestines to catalyze biological reactions which results in the breakdown of large food molecules to small building blocks which can be readily use by the body (CH102Labs, 2005). One of the enzymes

that is needed for digestion is the salivary amylase found in human saliva. Salivary amylase produced by salivary glands is a type of hydrolases enzyme. This type of enzyme catalyzes hydrolysis reactions by adding water to a bond which results to bond breakage. The substrate for salivary amylase is starch which is a polysaccharide and gives a product of maltose a disaccharide when acted by the enzyme amylase (Paine et al., 2010). Effectiveness of enzyme were influenced by several factors, including enzyme concentration, substrate concentration, pH, temperature, and presence of heavy metal cations which tests the activity of the enzyme (CHEM 1021, 2010). In the experiment performed human saliva was used which contains salivary amylase. Its enzyme activity was tested and specificity was determined under different temperature and pH although the levels of amylase vary considerably from one person to another. The objective of the experiment was to determine the optimum activity of salivary amylase by using different range of pH and temperature and examine its activity and specificity by altering temperature and pH.

Figure 1. Enzyme activity

of the mixture was dropped in the first well of the spot plate while two drops for iodine solution. This was the zero minute. After one minute interval three drops of mixture was added in the second well while iodine solution was dropped twice. This was the one minute. The previous steps were repeated until yellow-colored solution was observed. For other pH at 5, 6, 7, 8, AND 10 the whole step was repeated following the desired buffer. Then the reciprocal of time (1/t, min-1) against pH was plotted.

Results and Discussion

Materials and Methods

In this experiment the temperature and pH effect on the activity and specificity of salivary amylase was observed by testing it to different temperature and pH range. Except for enzyme solution which was prepared from the previous experiment, all the materials and reagents were provided by the laboratory. Effects of Temperature First, 2mL of enzyme solution was charged in a test tube and was labeled as 4C. Next, in a separate test tube 2mL of buffered starch solution was added and the test tubes were immersed and incubated in an ice bath for ten minutes maintaining the tem perature at 4C. After that the test tubes were immediately mixed and quickly the three drops of the mixture was dropped in the first well of the spot plate while two drops for iodine solution. This was the zero minute. After one minute interval three drops of mixture was added in the second well while iodine solution was dropped twice. This was the one minute. The previous steps was repeated until yellow-colored solution was observed. For the temperatures at 27C, 37C, 50C, 60C and 70C the whole step was repeated following the desired temperature. Then the reciprocal of time (1/t, min-1) against Temperature was plotted. Effects of pH First, mix 1 mL of acetate buffer (pH 4) and 1 mL of 2% unbuffered starch in a test tube then 2mL of enzyme solution was charged in a separate test tube. Next, the test tubes were immersed and incubated for ten minutes in a 37C water bath. After that the test tubes were immediately mixed and quickly the three drops

After performing the experiment the following data were obtained:

Time

Temperature 1/time 4C 0 6 27C 0.166667 2 37C 0.5 5 50C 0.2 60C 0 70C 0

Table 1. Effect of temperature Based on the table above it showed that temperature at 4C, 60C and 70C the reaction time needed for the reaction with iodine to give yellow solution was longer (affinity) compared to temperature at 27C, 37C and 50C. Plotting this result will give:
1/t (min-1) versus Temperature

0.6 0.5
1/t (in minutes)

0.4 0.3 0.2 0.1 0 20 40 60 temperature in C

1/t (min-1) versus Temperatur e

-0.1 0

80

Graph 1. 1/t vs T The graph above exhibited a Gaussian curve as a function of temperature. This graph showed that the peak of the graph corresponds to the

optimum temperature for the enzymatic activity of salivary amylase which was 37C.

Reaction Mechanism: starch + I2 -> blue/black complex

Time 3 2 1 3

pH

1/t 4 0 5 0.333333 6 0.5 7 1 8 0.333333 10 0

starch + Amylase -> maltose maltose + I2 -> no color change This was also the same as the effect of pH which gave an optimum pH at 7 since the starch at this pH was already been destroyed. At this optimum temperature and pH saliva amylase was not inhibited or degraded therefore enhanced the reaction process. In the case of the effect of temperature too much boiling would cause the molecules of the enzyme to vibrate to different degree depending on the temperature applied. If it is too high or too low the enzyme tend to change its 3D conformational structure by either making it ineffective or inhibiting the activity of enzyme. In terms of pH, different factors may affect the pH activity relationship of enzyme. These factors were the binding of the enzyme to substrate, catalytic activity of enzyme, ionization of substrate and variation of protein structure (Voet et al., 2008)

Table 2. Effect of pH Based on the table above it showed that at pH 4 and 10 the reaction time needed for the reaction with iodine to give yellow solution was longer (affinity) compared to pH at 5, 6, 7 and 8. Plotting this result will give:
1/t (min -1) versus pH

1.5
1/time (in minutes)

1
1/t (min -1) versus pH

0.5 0 0 5
pH

10

15

Graph 2. 1/t vs pH The graph above exhibited a Gaussian curve as a function of pH. This graph showed that the peak of the graph corresponds to the optimum pH for the enzymatic activity of salivary amylase which was at pH 7. The optimum temperature for saliva amylase was at 37C because based on the data obtained at this temperature the rate of reaction was fastest in converting starch to sugar when iodine was reacted to starch. This was also shown by the disappearance of the blue-black coloration in the mixture when iodine was added (see Figure 2). This occurred due to the activity of enzyme, salivary amylase which has already destroyed the structure of the starch (presence of yellow solution). Figure 2. Reaction of Iodine with starch with the presence of salivary amylase (catalyst)

Conclusions
Enzymes have optimum temperature and pH range. Deviation from this range may cause to decrease the effectiveness of the enzyme. At extreme conditions of temperature and pH would generally result in complete loss of activity for most enzymes.

At very low temperature or pH enzyme activity was inhibited, while increasing the temperature and pH at its optimum range it would enhance the reaction but exceeding at its threshold temperature and pH enzymes tend to change their conformational structure which was important to the function of many enzymes. Since the enzyme at optimum temperature and pH functions at its best, reaction of starch with iodine with the the presence of catalyst would eventually breakdown all of the starch and the reaction will not give the dark iodine/starch complex (CH102Labs, 2005). Over all reaction mechanism: POLYSACCHARIDE + SALIVARY AMYLASE -> MALTOSE + SALIVARY AMYLASE + smaller polysaccharides

of Starch using Salivary amylase. Retrieved on February 16, 2014.

References
Voet, D.J., Voet, J.G., and Pratt, C.W., rd (2008). Principles of Biochemistry,3 Ed. USA: Wiley & Sons, Inc. Paine, M.C., Olanka, N., Napa, J.M.J., Mendoza, E.V. (2010). Enzymatic Activity of Salivary Amylase. http://spot.pcc.edu/~mdeming/102/Labs/CH102_ Lab_9_Saliva_Amylase.pdf. Salivary amylase: An enzyme at the tip of your tongue. Retrieved on February 16, 2014. https://www.apsu.edu/sites/apsu.edu/files/chemi stry/SP11_1021_BREAKING_DOWN_STARCH _USING_SALIVARY_ENZYMES.pdf. Hydrolysis of Starch using Salivary amylase. Retrieved on February 16, 2014. Figures: Figure 1: https://www.apsu.edu/sites/apsu.edu/files/chemi stry/SP11_1021_BREAKING_DOWN_STARCH _USING_SALIVARY_ENZYMES.pdf. Hydrolysis of Starch using Salivary amylase. Retrieved on February 16, 2014. Figure 2: https://www.apsu.edu/sites/apsu.edu/files/chemi stry/SP11_1021_BREAKING_DOWN_STARCH _USING_SALIVARY_ENZYMES.pdf. Hydrolysis