Determining Optimum Temperature and Ph for Enzymatic Reactions of Alpha Amylase

2157 Words Jun 24th, 2007 9 Pages
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
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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
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