Full version Determining Optimum Temperature And Ph For Enzymatic Reactions Of Alpha Amylase

Determining Optimum Temperature And Ph For Enzymatic Reactions Of Alpha Amylase

This print version free essay Determining Optimum Temperature And Ph For Enzymatic Reactions Of Alpha Amylase.

Category: Science

Autor: reviewessays 19 February 2011

Words: 1958 | Pages: 8

Enzymes lower the activation energy of chemical reactions but they themselves are not consumed or altered when doing so. These catalysts work best at optimum temperatures and pH’s. The temperature and pH at which the reaction occurs the quickest is the ideal condition for the enzymatic reaction.

Alpha amylase converts starch into glucose and when starch is combined with I2KI indicator a dark purple solution forms. As the enzyme breaks down the starch the absorbency will decrease. The absorbency is measured through the spectrophotometer which reads the transmittance of the wavelengths that pass through the solution. In order to determine the optimum temperature for the enzymatic reaction water bath of varying temperatures were made. To determine the ideal pH various buffers were added to the solutions in order to adjust the pH.

At scheduled intervals the reacting solution was added to a cuvette containing I2KI indicator in order to stop the reaction, allowing for accurate readings of absorbency. Once completed graphs which compared absorbency to temperature and pH were created allowing for a visual reference of the results. Optimum temperature and pH were shown determined by noting where absorbency was the lowest because this is where the enzyme catalyzed the most amount of starch. The results showed that alpha amylase most efficiently converts starch into glucose at a temperature of 65C and pH of 5. Therefore, alpha amylase works best in mildly acidic conditions that have a high temperature.

Introduction

This experiment tested the cause and effect of environmental factors on enzymes reaction rate. The two environmental factors studied where temperature and pH. Enzymes are catalysts that lower the activation energy of specific reactions without themselves being consumed are altered in any way. When the activation energy is lowered it enables the reaction to be accelerated with less energy expenditure (Campbell, 2002). The rate is determined by speed at which a substrate binds to the enzyme to form an enzyme-substrate complex then decomposes to form the product. This speed is also determined by the concentration of the substrate, if the substrate concentration is higher then the possibility of forming an enzyme-substrate complex is also increased (Vliet, 2007).

The rate of enzymatic activity is based greatly on the surrounding environment. Catalytic activity occurs the most at optimum temperatures and pH’s which are specific to each enzyme. Increased temperatures increases molecular motion which in turn allow for increased substrate-enzyme collisions and more rapid reactions. But if the temperature is too high the enzyme is at risk for becoming denatured. Denaturization is an alternation in the tertiary structure of the enzyme which does not allow for efficient product conversions. This same possibility is likely to occur when the optimum pH is not met (Vliet 2007). Neutral environments have a pH of around 7 (Campbell, 2002). So if the environment is too acidic or basic then the enzymatic reaction rate is lowered.

This particular experiment studies the effect of temperature and pH on the enzyme alpha-amylase. Alpha amylase hydrolyzes starch into glucose molecules (Vliet, 2007). The reaction rate of the enzyme will be measured by determining the absorbency of the starch substrate. Since starch and iodine form a dark purple/blue substance the concentration of the solution allows us to determine the rate at which alpha amylase is converting starch into glucose. Also, an iodine indicator stops the reaction, which makes it possible to read the absorbency at a specific time interval. Absorbency is measured through a spectrophotometer which reads the amount of light that is being transmitted through a solution at a set wavelength (Vliet, 20007)

The purpose of this lab was to determine the optimum temperature and pH for which the enzyme alpha-amylase is most effective at. I predict that catalytic activity will be the most rapid at the 45C temperature because that is a normal body temperature. I also assume that an ideal pH for the reaction will be at 7 because it is a neutral pH.

Materials and Methods

A blank cuvette was created in order to set the spectrophotometers absorbance to zero. Contained in this cuvette was 5mL of distilled water and 0.1mL of I2KI indicator. This was used to recalibrate the spectrophotometer between the temperature and pH trials (Vliet, 2007).

To determine the most favorable temperature for the reaction of alpha amylase’s enzymatic conversion of starch to glucose water baths of various temperatures were used. First a stock solution was prepared prior to the experiment; it included alpha-amylase, I2KI indicator and .0033 g/ml of starch. Thirty-five mL of this solution was then combined with 35mL of distilled water into an Erlenmeyer flask in order to create the reaction flask. The flask was then added into one of the various water bath temperatures, 15, 35, 45, 55, 65 and 70 C. The reaction flask needed to be in the water baths long enough for it to reach the required temperature before the enzyme was added. 0.1mL of I2KI indicator was then added to each of the twelve cuvettes which were used in the timed readings. Then 5mL of the solution from the reaction flask was transferred into one of the cuvettes which contained I2KI indicator. Next, the starch-iodine complexes’ absorbance were read on the spectrophotometer and recorded onto the appropriate table. Then, 1mL of alpha amylase was added into the reaction flask to begin the reaction, once this step was complete the timing begun. Five mL of the solution was added from the reaction flask to a cuvette containing the I2KI indicator and the absorbency was read then recorded onto the appropriate table at one minute intervals. In order to be more precise the solution was stored in the pipette about 15 seconds before the time interval arrived so it could be released at the exact second required (Vliet, 2007).

To determine the optimum pH for the reaction stock solution was prepared prior to the experiment; it included alpha-amylase, I2KI indicator and .0033 g/ml of starch. Thirty-five mL of this solution was then combined with 35mL of the specified buffer into an Erlenmeyer flask in order to create the reaction flask. The various buffers used had pH’s of 4.0, 4.5, 5.0, 5.5, 6.0 and 6.5. Next, 0.1mL of I2KI indicator was then added to each of the twelve cuvettes which were used in the timed readings. Then 5mL of the solution from the reaction flask was transferred into one of the cuvettes which contained I2KI indicator. Next, the starch-iodine complexes’ absorbance were read on the spectrophotometer and recorded onto the appropriate table. Then, 1mL of alpha amylase was added into the reaction flask to begin the reaction, once this step was complete the timing begun. Five mL of the solution was added from the reaction flask to a cuvette containing the I2KI indicator and the absorbency was read then recorded onto the appropriate table at two minute intervals. In order to be more precise the solution was stored in the pipette about 15 seconds before the time interval arrived so it could be released at the exact second required (Vliet, 2007)

If the absorbance readings are greater then 0.8, then the absorbance can be calculated by noting the transmittance scale reading and incorporating it into the following equation: (Vliet, 2007)

A= 2-log10%T

Two graphs were then created to determine the best fit curve for each temperature and pH. The two figures needed were Absorbance vs. Time in regards to temperature and Absorbance vs. Time in regard to pH. The graphs allowed us to calculate the reaction rate by determining the best fit curve for each temperature and pH. Next, calculation needed to be made in order to determine the reaction rate for both temperature and pH. To find the change in absorbance, subtract the initial absorbance from the final absorbance. To find the numerator of the reaction rate equation divide the change in absorbance by 2. Then subtract the initial absorbance from the result just found. This number should be found on the absorbance axis, and then draw a horizontal line from that point to the best fit curve. To determine the time draw a line from the point just found on the best fit curve and drop it down vertically to the x-axis. Plug this value into the denominator of the reaction rate equation. Once the reaction rate is found two more graphs were made that compared the reaction rates of both temperate and pH (Vliet, 2007). The reaction rate equation is as follows:

Reaction Rate = ________Ð…Δ Absorbance________

Time that Ai – Ð…Δ Absorbance Took

Results

As the data shows when comparing absorbance vs. time in regards to temperature the optimum temperature is 60C and the reaction rate is around .25 An outlier existed at 65C but it was not included when the optimum temperature was determined, there a reanalysis shows the optimum temperature is around 60C. The absorbance vs. time in respect to pH showed that the largest reaction rate is 2.0 therefore the optimum pH is 5.

Table 1

Reaction Rate Calculations for Temperature

Temp. (C) Initial Absorbance

(Ai) Final

Absorbance

(Af) Initial – Final (ΔA) Ð… ΔA * Ai – Ð… ΔA TAi – Ð… ΔA ^ R.R (*/^)

15 1.15 .31 .84 .42 .73 .74 .06

35 .96 .17 .79 .395 .565 4 .10

45 1 .04 .96 .48 .52 2.2 .22

55 .89 .05 .84 .42 .47 1.7 .25

65 .95 .05 .9 .45 .5 1.2 .35

70 .73 .13 .6 .3 .43 1.6 .19

Table 2

Reaction Rate Calculations for pH

pH Initial Absorbance

(Ai) Final

Absorbance

(Af) Initial – Final (ΔA) Ð… ΔA * Ai – Ð… ΔA TAi – Ð… ΔA ^ R.R (*/^)

4.0 1.22 .66 .56 .28 .94 3.5 .08

4.5 1.22 .24 .98 .49 .73 3.3 .15

5.0 1.3 .05 1.25 .625 .68 3.3 .19

5.5 1.3 .09 1.21 .605 .57 3.0 .20

6.0 1.3 .38 .92 .46 .84 6.3 .07

6.5 1.3 .37 .93 .465 .835 6.2 .08

Discussion

Ideally the reaction between the enzyme alpha amylase and starch takes place at a temperature of 60C and at a pH of 5. At this specified temperature molecular motion is at its greatest speed. Increased speed resulted in more enzyme and substrate collisions thus the greatest enzymatic reactions. In conjunction with the ideal temperature, the optimum pH of 5 means that this reaction occurs best in acidic environments. At this optimal pH the hydrogen bonds are the least unaltered in comparison to the other pH’s. Having nearly unaltered hydrogen bonds the tertiary structure remains consistent. This allows for the enzyme to remain intact allowing it to continue converting substrates into products. Therefore the greater the reaction rate the more optimum the conditions because the enzyme alpha amylase is able to convert the substrate starch into glucose the quickest with the least energy expenditure.

It is assumed that the fastest reaction rates occur at optimum temperatures and pH’s. Other reactions had slightly slower reaction rates because they probably did not have extreme differences in the determining factors, temperature and pH. The most extreme conditions were probably at temperature of 15C and at pH of 4. In this case the enzyme was denatured causing the tertiary structure to be altered.

An error was evident in this experiment as there was a significant outlier in the temperature experiment. This could have arose because of several factors, most notably the enzyme could have been placed into the reaction flask before it reached the appropriate temperature or the reaction could have been stopped at a wrong time interval. Both could be prevented by being attentive to the experiment and reactions.

I was surprised to find that alpha-amylase was most effective at such a high temperature. Most enzymes are optimal around 35-40C. This would mean that alpha amylase is thermophilic because it is most effective at high temperatures. I also found it interesting that the pH was relatively acidic which would have me assume it is necessary in digestion. Digestive enzymes are much more acidic then neutral enzymes that have neutral pH’s of around 7 (Campbell, 2002). Therefore, my new hypotheses would suggest that alpha amylase is a digestive enzyme that has the highest enzymatic reactions at higher temperatures and lower pH’s.

Literature Cited

Campbell, N.A. 2002. Biology, 6th ed. Benjamin Cummings, San Francisco, CA

Vliet, K.A. 2007. A Lab Manual for Integrated Principles of Biology: Part One

– BSC 2010L, 2/e. Pearson Custom Publishing, United States