THE EFFECT OF VARYING TEMPERATURES AND p H’ s ON THE RATE OF REACTION OF AMYLASE FROM PORCINE PANCREATIC TISSUE

SOLAIMAN NAWAB

Major: Neuroscience

Class of 2023

Introduction

Enzymes are proteins that act as catalysts, accelerating the rate of a reaction by binding small molecules, called substrates, to their active site.1 Amylase is an enzyme that hydrolyzes, or breaks down, the substrate starch into the disaccharide maltose.2 Amylase is important to many organisms; for example, amylase helps the maturation of germinating seeds, since their embryos require a great deal of energy for growth and development.2 Additionally, porcine pancreatic amylase, the amylase selected for this experiment, plays a vital role in the digestion of feed in pigs; it breaks down starch with the addition of water into maltose, which is further broken down in order to become usable energy. Amylase was the first enzyme to be discovered in 1833 by French chemist Anselme Payen.3 In recent years, amylase has held an important role in the production of ethanol — to break starches in grains into fermentable sugar, producing shorter chains of oligosaccharides for various use.3

In order to thrive, enzymes must maintain specific environmental conditions, such as enzyme concentration, temperature, and pH. An enzyme at optimal environmental conditions will maximize the rate of its enzymatic reaction. The higher the enzyme concentration, the faster the rate of the reaction and vice versa. If the temperature is too hot, then the enzyme will denature. Colder temperatures reduce kinetic energy and slow the rate of the reaction.2 A much higher or lower pH both result in the denaturation of the enzyme. An enzyme that is denatured loses its specific threedimensional shape and structure, resulting in a loss of function and decline in enzyme activity. Since the amylase in this experiment was sourced from porcine pancreatic tissue, we therefore hypothesized the optimal environmental conditions for the amylase to have an undiluted concentration, a temperature of 102℉, and a pH of 7.5, based on the average internal body temperature of a pig and the alkaline bile produced by the pancreas as well as the large amounts of bicarbonate present in pancreatic juice.4 The purpose of this experiment was to understand how enzyme concentration influences the production of product and to understand the influence of temperature and pH on enzyme activity.

METHODS & MATERIALS

The experiment measured how the rate of an enzymatic reaction would be affected by manipulating the following variables: enzyme concentration, pH, and temperature. In these three subsections of the experiment, the protocol for the kinetic that measured the starch depletion was the same: after the enzyme was combined to react with starch, 2mL of the reaction mixture was removed, added to a test tube containing 3 drops of reagent, mixed via vortex mixer, transferred to a cuvette, and put into the spectrophotometer to record the absorbance of starch. This process was repeated every 30 seconds for 6 minutes total for each subsection. The only exceptions are subsections 2 and 3, which only recorded the absorbance in 1-minute intervals for a total of 7 minutes. Additionally, it is important to note that the spectrophotometer was set to a constant wavelength of 540 nm and the reagent used was kept constant as Lugol’s iodine.

In the first section, 3 Erlenmeyer flasks were prepared with 10mL solutions of undiluted enzyme, 1:3 dilution, and 1:9 dilution. In each flask, 20mL of starch was added to cause a reaction and the protocol for the kinetic was followed. This process was repeated for each of the 3 flasks. Additionally, the spectrophotometer was zeroed for each flask by using a cuvette of 1mL of the respective enzyme solution, 1mL of water, and 3 drops of Lugol’s iodine.

In the second section, 4 Erlenmeyer flasks were prepared to have pH’s of 4, 5, 6, and 7 and contain 2mL of starch solution. In each flask, 1mL of enzyme solution was added to cause a reaction and the protocol for the kinetic was followed. This process was repeated for each of the 4 flasks. Additionally, the spectrophotometer was zeroed for all 4 flasks by using 2mL water and 3 drops of Lugol’s iodine.

In the third and final section, the following temperatures were evaluated: 4°C, 22°C, 37°C, and 95°C. 4 Erlenmeyer flasks were prepared by adding 22mL of water and 2mL of starch solution. Additionally, 1mL of enzyme solution of a constant concentration was added to 4 test tubes. Each of the 4 flasks was heated and/or chilled with one of the 4 test tubes to its respective temperature. In each flask, 1mL of enzyme solution was added to cause a reaction and the protocol for the kinetic was followed. This process was repeated for each of the 4 flasks. The spectrophotometer was zeroed for all 4 flasks by using 2mL water and 3 drops of Lugol’s iodine.

Results

In general, the trendlines of the absorbance graphs (Figures 1,3,5) are identical to the trendlines of their corresponding percent amylose graphs (Figures 2,4,6). This means that as the absorbance of starch from the spectrophotometer decreases over time, the percent amylose in the solution decreases over time as well. The decrease in the absorbance of starch and percent amylose from the spectrophotometer over time is due to the enzyme using up the starch during the reaction. Amylose is one of two main molecules that starch is composed of, making up 20% of starch. At first, there is a high percent amylose, as the reaction is just starting, and the starch is beginning to be consumed. However, as the reaction goes on, there is little starch remaining, leading to a decrease in percent amylose. It is important to note that the percent amylose in Tables 1-3 was calculated based on an amylose standard curve that was given.

The graph illustrates the absorbance of starch for the undiluted enzyme, 1:3 enzyme dilution, and 1:9 enzyme dilution. As time went on, the absorbance of starch read from the spectrophotometer decreased, with the undiluted enzyme showing the most prominent decrease.

The graph illustrates the percent amylose of the undiluted enzyme, the 1:3 enzyme dilution, and 1:9 enzyme dilution after starch was added. As time went on, the percent amylose calculated from the standard curve decreased, with the most prominent decrease shown from the undiluted enzyme.

Table

The graph illustrates the absorbance of starch for the enzyme at pH 4, pH 5, pH 6, and pH 7 after starch was added. As time went on, the absorbance read from the spectrophotometer tended to decrease, with the most prominent decrease at pH 7.

FIGURE 4: The Effect of pH on the Rate of Reaction Based on Percent Amylose

The graph illustrates the percent amylose of the enzyme at pH 4, pH 5, pH 6, and pH 7 after starch was added. As time went on, the percent amylose calculated from the standard curve tended to decrease, with the most linear decrease at pH 7.

TABLE 3: The Absorbances and Percentages of Amylose at Various Temperatures

FIGURE 5: The Effect of Temperature on the Rate of Reaction Based on Absorbance

The graph illustrates the absorbance of starch for the enzyme at various temperatures after starch was added. As time went on, the absorbance read from the spectrophotometer decreased, with 22°C having the most prominent decrease.

FIGURE 6: The Effect of Temperature on the Rate of Reaction Based on Percent Amylose

The graph illustrates the percent amylose of the enzyme at various temperatures after starch was added. As time goes on, the percent amylose of the solutions decreases, with 22°C having the most prominent decrease.

The purpose of this experiment was to understand how enzyme concentration influences the production of product and to understand the influence of temperature and pH on enzyme activity. The enzyme used in this experiment was amylase from porcine pancreatic tissue. The experiment was carried out by adding starch solution to the amylase at varying concentrations, temperatures, and pH’s and measuring the absorbance of starch by a spectrophotometer. The anticipated outcome for the optimal environmental conditions of amylase was an undiluted concentration, a temperature of 102℉, and a pH of 7.5, based on the average internal body temperature of a pig and the alkaline bile produced by the pancreas as well as the large amounts of bicarbonate present in pancreatic juice.4

The data supported the undiluted concentration to be the most effective concentration at maximizing enzyme activity based on the drastic decrease in percent amylose (0.0241 to 0.0071) and starch absorbance (1.51 to 0.44) relative to the other concentrations in Table 1. The hypothetical optimal pH of 7.5 was slightly inaccurate since the data supported a pH of 7 to be the most effective at maximizing enzyme activity. This is based off pH 7 showing the greatest decrease in percent amylose (0.0066 to 0.0033) and starch absorbance (0.41 to 0.01) relative to the other pH’s in Table 2. The data did not support the hypothetical optimal temperature to be 102℉ (38.9°C), as 22°C illustrated the most prominent decrease in percent amylose (0.0077 to 0.0011) and starch absorbance (0.48 to 0.06) relative to the other temperatures in Table 3.

Further study is required in order to determine why the optimal temperature of the amylase was determined to be 22°C instead of 102℉ (38.9°C). It is possible that error may have occurred since Table 3 was based off data that was provided instead of personally gathered in the experiment. Additionally, another study that used amylase from porcine pancreatic tissue found the optimal temperature for enzyme activity to be 50°C.5 This signals that discrepancy when determining the optimal temperature of an enzyme may be more common that previously thought. Further study is required in enzymes, porcine pancreatic amylase, and the environment of the pancreas itself. The results found in this experiment could give more insight into human digestion as well as provide guidelines for improving the efficiency of industrial ethanol production.