Reaction Rates of Barley Alpha Amylase with Starch at Specific Ph's and Temperatures

Reaction Rates of Barley Alpha Amylase with Starch at Specific pH's and Temperatures

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Abstract

Enzyme's are used as catalysts in certain reactions. They help lower the activation energy needed for the reaction to go to completion. At optimum temperature and pH the amount of collisions of substrate and enzyme is at its highest, any deviation from the optimum temperature and pH will result in the denaturization of the enzyme. The purpose of this experiment is to find the optimum temperature and pH for the reaction of Barley alpha-amylase and starch. The predicted optimum temperature and pH is 50-60 degrees Celsius and pH of 5-6. To test this hypothesis, a starch solution was mixed with the amylase and then tested for absorbance, using a spectrophotometer, at several different temperatures and pHs. From these experiments it was concluded that the optimum temperature was 55 degrees Celsius and the optimum pH was 5. these results lead to the conclusion that the Barley seed is best suited to be in soil that is relatively acidic and warm.

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Introduction

The purpose of this enzyme kinetics lab was to experimentally find the optimum pH and temperature at which the enzyme, Barley alpha-amylase, would react with a starch solution. An enzyme is a special protein that is used to catalyze certain reactions (Campbell, 2005). A catalyst is a substance that speeds up a reaction without being consumed by the reaction (Campbell, 2005). Reactions are measured by their rate or reaction. The reaction rate can be increased and decreased based on the amount of activation energy is applied to it. The purpose of the enzyme is to lower this activation energy to create product more efficiently (Campbell, 2005). When an enzyme binds with a substrate it forms an enzyme-substrate complex. This complex is how the enzyme reacts with the substrate to form the product, such as when the barley alpha amylase bonds with the starch to form barley alpha amylase, maltose, and glucose (Vliet, 1996). The enzyme remains relatively unchanged within the enzyme-substrate complex while the substrate is broken down into product. Two factors that influence the enzyme-substrate complex are the frequency of formation of the enzyme-substrate complex and the time it takes the enzyme to react with the substrate (Vliet, 1996). The frequency of reaction is dependent on the amount of substrate in the reaction. As the reaction takes place there will be less and less substrate for the enzyme to react with, therefore reducing the frequency of the formation of the enzyme-substrate complex (Campbell, 2005). There are also factors within the physical environment that can affect the catalytic activity of the enzyme.

The two main environmental factors that affect enzymes are the pH and the temperature of the reaction. When a reaction is heated the molecules with in it tend to speed up, which

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increases the chance of enzyme-substrate connection (Vliet, 1996). The pH of the reaction also is an important factor in the formation of the enzyme-substrate complex. IF the temperature or pH of the reaction are higher or lower than that of the optimum the enzyme's structure can become denatured (Vliet, 1996). If this happens the enzyme will not be able to correctly bind with the substrate to form the product.

The enzyme, alpha amylase, is commercially harvested from the Barley seed (Vliet, 1996). The developing seeds use this enzyme to hydrolyze starch into glucose (Vliet, 1996). With the reaction of this enzyme and starch the reaction rate was measured at specific temperatures and pH's by using a spectrophotometer, a machine that measures the absorbance of solutions by passing light through a sample of the solution (Vliet., 1996). As time goes on the enzyme breaks down the starch within the solution and lowers the absorbance reading. Using this data a calculation can be made concerning the reaction rate of the alpha-amylase and starch reaction. Also the optimum pH and temperature for this reaction can be found.

The optimum temperature and pH for the reaction of alpha-amylase and starch should be 50-60 degrees Celsius and a pH of 5-6. Every reaction has an optimal temperature and pH. When the reaction is pushed above or below these optimal ranges the protein can become denatured through the weakening of its hydrogen bonds (Campbell, 2005). If the temperature is to hot or the pH is to acidic, then the enzyme will no longer be able to combine with the substrate due to the deformation of its structure (Campbell, 2005). At the optimal ranges the greatest number of collisions are occurring between the substrate molecules and the enzyme (Campbell, 2004). This allows for the most efficient formation of product.

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Materials and Methods

This experiment was separated into two parts: one section was testing the pH's affect on absorbance of the starch solution and the second was the affect of temperature on absorbance. The absorbance was measured using a spectrophotometer which was turned on and allowed to warm up for 15 minutes and then set to 560nm.

To begin the pH experiment, 12 cuvettes were filled with 0.1 mL of I_2_KI indicator. Using one of the 12 cuvettes, a "blank" was made by adding 5 mL of water to the 0.1 mL of the I_2_KI indicator. The "blank" was used to set the spectrophotometer absorbance reading to zero. Also before the readings were taken, 35 mL of the stock starch solution (0.0033 g/mL) and 35 mL of the pH 5.0 solution where mixed in a reaction flask. Before the alpha-amylase was added 5mL of the solution in the reaction flask was added to one of the cuvettes and the time zero reading was recorded. Once this was done 1 mL of alpha-amylase was added to the reaction flask and timing was begun. Every two minutes, for a total of 20 minutes, after the amylase was added 5 mL of solution was transferred to a cuvette and the reading was taken. The spectrophotometer was blanked between each reading. This procedure was performed for 5 other pH solutions: 4, 4.5, 5.5, 6.0, and 6.5.

To begin the temperature experiment, another set of 12 cuvettes were filled with 0.10 mL of I_2_KI indicator.