Determining Optimum Temperature and Ph for Enzymatic Reactions of Alpha Amylase

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